Antitumor and off-target effects of cholesterol-conjugated let-7a mimics in an orthotopic hepatocellular carcinoma xenograft nude mouse model : Journal of Bio-X Research

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Antitumor and off-target effects of cholesterol-conjugated let-7a mimics in an orthotopic hepatocellular carcinoma xenograft nude mouse model

Guan, Jiana,*; Liu, Mingyangb; Li, Xinc,d; Zhou, Liangruib; Dong, Xueyue; Dai, Weif; Xia, Yug; Yang, Taoa,b; Guo, Shaojuana; Li, Xingqib; Han, Yehuaf; Luo, Yufengb

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Journal of Bio-XResearch 5(4):p 181-196, December 2022. | DOI: 10.1097/JBR.0000000000000103

Abstract

Introduction

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer mortality.[1] It is the second leading cause of cancer-related death to lung cancer in China.[2] There is a high recurrence rate of HCC after surgery, and chemotherapy and radiotherapy have limited efficacy for HCC and are associated with serious toxicity.[1,3] Thus, new therapeutic strategies for HCC are urgently needed, especially for advanced-stage patients.

MicroRNAs (miRNAs) are endogenous, non-coding, small RNAs that repress gene expression at the post-transcriptional level by base pairing to the 3'-untranslated region of target messenger RNAs, and are important mediators of carcinogenesis and predictors of clinical prognosis.[4–6] Functional studies have confirmed that miRNAs act as tumor suppressors and are dysregulated in many types of cancer. Thus, modulating miRNAs represents a promising new tool for cancer therapy.[7,8] The let-7 family of miRNAs function as typical miRNA tumor suppressors in their ability to target multiple mRNAs, which makes them interesting candidates for development as miRNA mimic-based therapeutics.[9]

In recent years, both viral and nonviral delivery vehicles have been developed for the delivery of RNA, including the miRNA gene or mimics in vivo.[10,11] However, both types remain challenges for potential clinic use in the future. As an example of viral delivery, the adeno-associated virus (AAV) vector for miRNA delivery has low efficiency in terms of miRNA expression, and provokes an immune reaction in response to the viral proteins, and shows dose-dependent toxicity in patients, all of which pose significant hurdles to its potential clinical use. In addition, AAV-mediated recombinant miRNA gene expression in target cancer cells decreases rapidly as the non-replicating episomal viral genome is lost from the cells.[12] Nonviral vehicles that have been tested in vivo involve the use of miRNA mimic modifications or carriers that facilitate release from endosomes, thereby improving delivery to target cells and potentially increasing the therapeutic effects. Using nonviral delivery vehicles, miRNA mimics, such as those mimicking the miR-122 and let-7 miRNA families, have shown effective anti-HCC efficacy in vivo in preclinical studies.[13,14]

Among the delivery system, some RNA-based drugs are modified with 3′ cholesterol that can aid the association of the siRNA to lipoprotein particles, lipoprotein receptors and transmembrane proteins, and thus target the siRNA to specific tissues, such as the liver.[10] In addition, as an essential component of animal cell membranes, and, cholesterol is an efficient transmembrane transporter and considered non-toxic. We previously showed that cholesterol-conjugated let-7a (Chollet-7a) exerted significant antitumor effects by down-regulating all three human ras genes in vivo, including n-ras, k-ras, and h-ras. However, our in vitro experiments also suggested that cholesterol-conjugated miRNA mimics have potential off-target effects, as indicated by morphological differences between cells treated with a negative control miRNA mimic (Chol-miRCtrl) and untreated tumor cells. Compared to untreated (blank) tumor cells, the Chol-miRCtrl-treated cells exhibited decreased survival and viability.[15] The potential off-target effects that may occur in vivo remain unknown; and it is crucial for off-target effects to be minimized when using this approach. In this study we investigated the antitumor efficacy and potential off-target effects of cholesterol-conjugated let-7a miRNA delivered by either a continuous or an intermittent dosing regimen, to provide important information for the future study and potential clinical use of miRNA mimics.

Materials and methods

Experimental animals and miRNAs

All in vivo experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and the experimental animal welfare ethics regulations of China, with the approval of the Institutional Research Board, Peking Union Medical College Hospital. All animal experiments were performed at the Centre for Experimental Animal Research (SPF cleanliness, license No. SYXK (Jing) 2010-0021, Beijing, China), Institute of Basic Medical Sciences (IBMS), CAMS & PUMC.

BALB/C nude (nu/nu) mice (6-7 weeks old, 20 ± 3g) were purchased from the National Institutes for Food and Drug Control (lot No. 11400500001092; Beijing, China). The cholesterol-conjugated let-7a (Chol-let-7a) and negative control miRNA (Chol-miRCtrl) mimics were purchased from Ribobio (Guangzhou, China).

Orthotopic xenograft nude mouse model

HepG2 cells (2 × 106) were injected directly into the livers of 20 nude mice. One week later, the formation of HepG2 orthotopic xenografts was confirmed by ultrasonography using a Vevo 2100 high-frequency ultrasound system (VisualSonics, Inc. Beijing, China). Fifteen successfully engrafted mice were randomized into three cohorts of five animals.

Assessment of antitumor and off-target effects of cholesterol-conjugated molecules administered continuously or intermittently

Two dosing regimens were designed to evaluate the antitumor efficacy and off-target effects of Chol-let-7a and Chol-miRCtrl.

We first evaluated the effects of continuous delivery of Chol-let-7a on HepG2 orthotopic xenografts in nude mice.'10' The mice were injected with Chol-let-7a or Chol-miRCtrl (5nmol in 250 μL saline) via the tail vein every 3 days for 5 weeks, after which the tumor size (volume, mm) and changes in liver and kidney tissues and cells were observed. Xenograft mice injected with the same volume of 0.9% physiological saline were used as the negative xenograft control (blank) group.

To observe the effects of the cholesterol-conjugated molecules on normal mice in the absence of tumor, 10 nude mice were randomized to two groups of five animals each and injected with Chol-let-7a or Chol-miRCtrl according to the dosing regimen described above.

To assess the effects of intermittent dosing, mice with orthotopic xenografts and normal nude mice were injected with Chol-let-7a, Chol-miRCtrl, or saline as above every 3 days for 2 weeks, left uninjected for 1 week, and then injected every 3 days for another 2 weeks.

At the end of the 5-week experimental period for all groups, tumor size was measured on three orthogonal axes (a, b, and c), and tumor volumes (mm3) were calculated according to the following formula: V = (abc)/2.[16] Samples of tumor, liver, kidney, and other tissues from all animals were harvested and snap-frozen or preserved in 10% neutral buffered formalin rapidly for further examination.

Histopathological examination under light microscope

All samples fixed in 10% neutral buffered formalin were routinely processed and embedded in paraffin; and were submitted for standard processing with hematoxylin and eosin staining (HE), Histopathological features was evaluated with HE sections observed under histopathological examination under the light microscope (LM) (OLYMPUS Corporation, Japan).

Transmission electron microscopy

Fresh and snap-frozen tissues were fixed with 2.5% glutaraldehyde for 30 minutes at room temperature, followed by 1.5 hours in 2% OsO4. Samples were stained and examined with a transmission electron microscope (JEOL JEM 1010, Tokyo, Japan), and digital images were obtained with an Erlangshen ES1000W camera (Model 785, Gatan, Warrendale, PA, USA).

In vivo whole-animal imaging and distribution analysis

Fluorophore-labeled synthetic Chol-let-7a or Chol-miRCtrl (RiboBio, Guangzhou, China) was administered via the tail vein. Two mice with HepG2 orthotopic xenografts were injected with Cy5-labeled Chol-let-7a, two mice with HepG2 orthotopic xenografts were injected with Cy5-labeled Chol-miRCtrl, two normal nude mice were injected with Cy5-labeled Chol-let-7a, and two normal nude mice were injected with Cy5-labeled Chol-miRCtrl. Whole-animal images were recorded at 15, 45, 60, and 100 minutes, and 19, 24, 48, and 72hours after treatment using the NightOWL LB 983 in vivo imaging system (Berthold, Beijing, China). Digital images were produced and analyzed with Indigo software (Berthold).

In vitro con foca and phase fluorescence microscopy

Two pairs of xenograft mice and two pairs of normal mice were injected with Cy5-labeled Chol-let-7a or Chol-miRCtrl (5nmol in 250 μL saline buffer). Fresh orthotopic tumor and liver tissues were snap-frozen 2 hours after injection of Chol-let-7a or Chol-miRCtrl. Frozen sections with a thickness of 10-20 μm were observed and imaged using a confocal laser scanning microscope (OLYMPUS FV1000, OLYMPUS Inc., Shanghai, China) and a phase fluorescence microscope (Leitz, Laborlux 12).

Quantification of let-7a miRNA expression

Total miRNA was isolated from snap-frozen HepG2 xenograft tumor and paracancerous liver tissue using a mirVANATM PARISTM RNA isolation kit (Applied Biosystems, Carlsbad, CA, USA). RNA (10 ng) was reverse-transcribed using a miRNA Reverse Transcription Kit (Applied Biosystems) and let-7a-specific primers (TaqMan miRNA Assay, Applied Biosystems).

Quantitative real-time PCR was performed with an ABI Prism 7500 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, USA) and the Perkin-Elmer Biosystems analysis software according to the manufacturer's instructions. RNU6B was used as a housekeeping control gene. Relative expression was calculated using the 2−∆∆CT method.[17]

Determination of serum cytokines

Twelve normal nude mice and twelve mice with HCC orthotopic tumors were injected with either Chol-let-7a or Chol-miRCtrl (n = 6 per group) every 3 days for 4 weeks, beginning 1 week after the HepG2 cells were implanted. Serum samples were obtained from the tail vein at the beginning and end of the 4-week experimental period, and the levels of four cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF) were quantified by multiplex assays. Cytokine levels (IL-6, IL-8, TNF-α, and VEGF) in blood serum samples (200 μL) were quantified with ELISA using a commercial kit (96-Well Plate MILLIPLEX® MAP, Merck Millipore, Darmstadt, Germany) and a Lunimex 200 instrument (MILLIPLEX®, Germany). The data were analyzed using Milliplex Analyst software.

Serum samples were obtained prior to and after Chol-let-7a or Chol-miRCtrl administration, and the levels of four cytokines were examined. The cytokine levels are shown in Additional Table 1 (https://links.lww.com/JR9/A31).

Table 1 - Liver histopathology and ultrastructural changes in normal and xenograft mice after treatment
Group Histopathological findings TEM features
Sinusoidal structure
Inflammation features Necrosis Sinusoidal dilation Congestion Inflammation features Irregular organelles Dilation Disrupted structures Cell hyperplasia†
Chol-let-7a-treated normal mice Mild Focal /spotty Diffuse Mild Mild (leukocyte) Yes Yes Yes, mild/less Yes
Chol-miRCtrl-treated normal mice Mild Focal /spotty Diffuse Mild/Severe Mild (leukocyte) Yes Yes Yes, mild/less Yes
Xenograft with no treatment Severe Severe Irregular Focal Severe Severe (leukocyte) No Mild Yes Yes
Cho/-lef-7a-treated xenograft Mild/severe Mild/severe Diffuse Mild/Severe Mild Leukocyte Yes Yes Yes Yes
Chol-miRCtrl-treated xenograft Severe Severe Diffuse Severe Severe (leukocyte and macrophage) Yes Severe Yes Yes
*Organelle abnormalities in parenchymal cells. Sinusoidal endothelial cell hyperplasia. TEM = transmission electron microscopy.

Statistical analysis

Data are expressed as the mean ± SEM. All data analyses were performed using SPSS 16.0 software (SPSS, Chicago, IL, USA). Student's t-test was used for statistical comparisons between groups. P < 0.05 was considered to be statistically significant.

Results

Antitumor efficacy of Chol-let-7a in orthotopic xenograft models

Orthotopic tumor sizes were evaluated at the end of 5 weeks of treatment with the continuous or intermittent dosage regimen (Fig. 1). The tumor size (volume, mm3) in the continuous-dosage Chol-let-7a-treated xenograft group was 152.15 ± 38.43, which was 35.86% (P < 0.01) and 40.02% (P < 0.01) the size of the tumors in the two control groups (Chol-miRCtrl, 424.34 ± 60.10; blank xenograft group, 380.23 ± 74.83). At the end of the intermittent treatment regimen, the tumor sizes in the Chol-let-7a-treated, Chol-miRCtrl-treated, and blank xenograft group were 163.66 ± 25.81, 250.16 ± 44.91, and 288.85 ± 38.59, respectively; the tumors in the Chol-let-7a treated mice were significantly smaller than those in the Chol-miRCtrl-treated and blank xenograft groups, at 65.42% (P = 0.012) and 56.66% (P = 0.031), respectively. There was no significant difference in tumor size in the Chol-miRCtrl and blank xenograft groups (P = 0.23).

F1
Figure 1.:
Orthotopic tumor growth and metastasis inhibited by systemic treatment with Chol-let-7a. To assess antitumor efficacy in vivo, we evaluated the effects of Chol-let-7a, administered via a continuous or intermittent dosage regimen, on HepG2 orthotopic xenografts in nude mice. Orthotopic tumor size was evaluated at the end of 5 weeks. Gross specimen analysis showed that both dosing regimens inhibited tumor growth (area in the yellow band) and metastasis. (A) Continuous dosing regimen. (B) Intermittent dosing regimen.

Effects of Continuous administration of Chol-let-7a or Chol-miRCtrl on the liver and kidney in xenograft and normal mice

LM and transmission electron microscopy analysis of hepatic tissues and cells

Hepatic tissues from xenograft mice and control mice were observed after continuous administration of Chol-let-7a, Chol-miRCtrl and saline only (blank) for 5 weeks. LM analysis (Fig. 2) showed diffuse, chronic, nonspecific inflammation in the liver parenchyma of all three xenograft mice. Spotty and focal necrosis and mass lesions/confluent necrosis were observed. The necrosis was milder in the Chol-let-7a-treated group than in the two xenograft control groups. Significant diffuse sinusoidal dilation and edema, primarily around the centrolobular vein, were observed in the Chol-let-7a-and Chol-miRCtrl-treated groups. Higher magnification showed sinusoidal endothelial cell hyperplasia in the Chol-let-7a-, and Chol-miRCtrl-treated xenograft mice; and significant centrolobular vein congestion and necrosis was predominantly observed in the Chol-miRCtrl and blank xenograft groups (Fig. 3A). In addition, focal irregular sinusoidal dilation with congestion was found primarily in the Chol-miRCtrl and blank xenograft groups, along with some leukocyte infiltration.

F2
Figure 2.:
Light microscope images of liver sections from xenograft and normal mice after continuous treatment with Chol-let-7a or Chol-miRCtrl. The images show focal necrosis and mass lesions in the xenograft mice, and diffuse sinusoidal dilation in the Chol-let-7a- and Chol-miRCtrl-treated xenograft mice and normal mice under hematoxylin-eosin staining. Scale bars = 100 mm.
F3
Figure 3.:
Liver histopathology and ultrastructural features after continuous therapy. (A) Light microscopy findings under high magnification. The images show dilated sinusoids. Also shown are sinusoidal endothelial cell hyperplasia (white arrows); mild focal and spotty apoptotic cells (yellow arrows); and inflammatory cells (orange arrows) under hematoxylin-eosin staining. Scale bars = 20 μm. (B) Transmission electron microscopy (TEM) images of hepatic sinusoidal structures. The images show edematous and dilated sinusoidal and perisinusoidal lumens with structural damage and mild fibrosis of the hepatic sinusoids (red arrows) in the Chol-let-7a- and Chol-miRCtrl-treated groups. Also shown are leukocytes (yellow arrows) in the Chol-miRCtrl-treated and blank control groups. Scale bars = 2 μm. (C) Ultrastructure of liver parenchymal cells after continuous treatment with Chol-let-7a (TEM). Here shows some organelle abnormalities, enlarged, irregular, swollen mitochondria (Mit); autophagic bodies (Aut), and multilamellar bodies (MLB) observed in the hepatocyte cytoplasm. Scale bars = 200 nm.

Sinusoidal and perisinusoidal lumen dilation in the hepatic tissue from Chol-let-7a- and Chol-miRCtrl-treated was confirmed by transmission electron microscopy (TEM) (Fig. 3B). The tissue samples from some of these mice exhibited disrupted hepatic sinusoid structure (3/5 Chol-let-7a-treated mice; 4/5 Chol-miRCtrl-treated mice) and hepatic sinusoid fibrosis (2/5 Chol-let-7a-treated mice; 3/5 Chol-miRCtrl-treated mice). In addition, some organelle abnormalities, such as enlarged, irregular, or swollen mitochondria, autophagic bodies, and multilamellar bodies, were observed in the hepatocyte cytoplasm (4/5 Chol-let-7a-treated mice; 4/5 Chol-miRCtrl-treated mice) (Fig. 3C).

The same experiment was performed in normal nude mice to exclude the effects of xenograft tumor growth. In hepatic tissue from these mice, diffuse sinusoidal dilation was observed after Chol-let-7a- and Chol-miRCtrl treatment. However, the inflammation and necrosis were significantly milder than in the xenograft mice (Fig. 2). LM analysis of the hepatic tissue showed chronic inflammation and fibrosis, along with nonspecific focal degeneration and necrosis or focal or spotty apoptotic hepato-cytes (Fig. 2). Discontinuous central lobular veins and bile ducts accompanied by non-specific reactive cell proliferation and inflammatory cell infiltration were occasionally observed.

LM and TEM analysis of renal tissues and cells

Renal tissues and cells from xenograft and normal mice treated continuously with Chol-let-7a or Chol-miRCtrl were then assessed by LM and TEM.

LM analysis of renal tissue showed mild inflammatory cell infiltration and interstitial alterations, such as intertubular blood capillary dilation and intertubular hemorrhage, in all three xenograft groups. These effects were relatively mild in the Chol-let-7a-treated group. Dilated glomerular lumens, renal cell dissociation, and renal tubule necrosis were seen in all three xenograft groups under high magnification. Focal segmental sclerotic glomeruli were occasionally observed in the blank xenograft mice. Congested glomeruli and intertubular blood capillary dilation were predominantly observed in the Chol-miRCtrl-treated and blank xenograft control mice. No significant congestion was observed in the Chol-let-7a-treated group. However, mild hypercellularity with dilated patent capillary lumens was observed in the Chol-let-7a- and Chol-miRCtrl-treated mice. Congestion and hypercellularity were observed in the Chol-miRCtrl-treated and blank xenograft mice.

The features described above were confirmed by TEM (Fig. 4). Congestion was primarily observed in the glomerular capillaries and proximal tubule interstitia in the Chol-miRCtrl-treated and blank xenograft mice; inflammatory cells were also more prevalent in the glomeruli in the two control groups. As shown in Figure 4A and B, some features were similar in the xenograft mice after Chol-let-7a and Chol-miRCtrl treatment. The features and their incidence rate in Chol-let-7a- and Chol-miRCtrl-treated mice were: glomerular features such as dilated lumens and endocapillary membrane hyperplasia (5/5, 5/5) and focal foot[5] process effacement and focal basement membrane thickening (3/5, 2/5). In addition, inclusion bodies (4/5, 5/5) were readily apparent in the glomerular capillary endothelial cells (Fig. 4A and B). The tubule cells exhibited abnormal mitochondria (Mit) (4/5, 4/5), multilamellar bodies (MLBs)[7] (3/5, 3/5), and autophagic bodies (Aut) (3/5, 4/5) (Fig. 4A and C). However, dilated and destroyed interstitial structures with inflammatory cell infiltration, as well as mild tubulitis, were observed in all three xenograft groups (Fig. 4A).

F4
Figure 4.:
TEM Images of the kidney ultrastructure after continuous treatment. Renal tissues and cells from xenograft and normal mice treated continuously with Chol-let-7a or Chol-miRCtrl were assessed by TEM. Compared to those in normal mice, congestion was primarily observed in the glomerular capillaries and proximal tubule interstitia in the Chol-miRCtrl-treated and blank xenograft mice; inflammatory cells were also more prevalent in the glomeruli in the two control groups. Some features were similar in the xenograft mice after Chol-let-7a and Chol-miRCtrl treatment. (A) Glomeruli and proximal tubules. The images show inclusion bodies in the glomerular endothelial cells (short red arrows), leukocytes (yellow arrows), and macrophages (orange arrow) in the interstititia, and incomplete interstitial structure (areas between two long thin red arrows). Scale bars = 2 μm. (B) Glomerular changes. Membrane hyperplasia in glomerular capillary cells with bridge structure (wide black arrow) and grid structure (thin black arrow); inclusion bodies (red arrow) in the endothelial cells (En); and focal podocyte (Po) foot process effacement (yellow arrows). Scale bars = 0.5 μm. (C) Changes in proximal tubule cells. Enlarged and irregular mitochondria with vacuoles (white arrows), irregular lysosomes (orange arrow), autophagy (blue arrow), and multilamellar bodies (yellow arrow). Scale bars = 1 μm. TEM = Transmission electron microscopy.

In normal mice, the congestion and inflammation were mild. However, hyperplasia of the glomerular endocapillary cell membrane (3/4, 3/4) and the presence of inclusion bodies in glomerular capillary endothelial cells were observed in Chol-let-7a- and Chol-miRCtrl-treated mice (3/4, 3/4) (Fig. 4A). Inflammatory cells were occasionally observed in the dilated glomerular lumen in Chol-let-7a and Chol-miRCtrl-treated normal mice (2/4, 1/4) (Fig. 4A).

The LM and TEM findings regarding off-target effects of this cholesterol-conjugated delivery system are summarized in Tables 1 and 2.

Table 2 - Kidney histopathology and ultrastructural changes in normal and xenograft mice after treatment
Group Histopathological findings TEM features
Glomeruli Tubule and tubular interstitia
Inflammation Congestion Dilated lumen of glomeruli Dilated lumen† Membrane hyperplasia† Inclusion bodies† Congestion Tubulitis Congestion Dilation Dilated Incomplete
Cho/7ef-7a-treated normal mice Mild Irregular, mild Yes Yes Yes, mild Yes Mild Mild Mild Mild
Chol-miRCtrl-treated normal mice Mild Irregular, mild Yes Yes Yes, mild Yes Mild Mild Mild Mild
Xenograft with no treatment Severe Severe Severe/focal Focal No No Severe Severe Severe Focal severe
Cho/7ef-7a-treated xenograft Mild/severe Mild Yes Mild Yes, mild Yes Mild Mild Mild Mild
Chol-miRCtrl-treated xenograft Severe Severe Yes Yes Yes, mild Yes Yes, severe Yes, severe Yes, severe Yes, severe
*Membrane hyperplasia of the glomerular capillary wall. Inclusion bodies in endothelial cells. Dilated and incomplete tubular interstitia. TEM=transmission electron microscopy.

Effects of intermittent administration of Chol-let-7a or Chol-miRCtrl on the liver and kidney in xenograft and normal mice

Next, we examined the effects of injection with Chol-let-7a, Chol-miRCtrl, or a saline treatment as blank control on the livers and kidneys of xenograft and normal mice, and compared the findings to those from mice that received the continuous dosing regimen.

Inflammation and mass necrosis were observed in the two control xenograft mouse groups (Chol-miRCtrl-treated and blank xenograft); mild inflammation and focal and spotty necrosis were observed in the Chol-let-7a-treated mice. Under TEM, organelle abnormalities were occasionally observed in the hepatic parenchymal cells in all xenograft mice. However, only mild, non-specificinflammation was observed in normal mice after injection of Chol-let-7a or Chol-miRCtrl.

Diffuse sinusoidal dilation and edema and mild sinusoidal endothelial cell hyperplasia were observed in the Chol-let-7a- and Chol-miRCtrl-treated xenograft and normal mice (Fig. 5A), but at milder levels than those observed in the mice that received the continuous dosing regimen.

F5
Figure 5.:
Changes in the liver and kidney after intermittent treatment with Chol-let-7a or Chol-miRCtrl. Mice with orthotopic xenografts and normal nude mice were injected with intermittent dosing of Chol-let-7a, Chol-miRCtrl, or saline (blank), and compared the effects of the molecules on hepatic and renal tissues and cells under light microscope (LM) and transmission electron microscopy (TEM). Here show the findings of histopathological and ultrastructures features in livers and kidneys under LM and TEM to those from mice that received the continuous dosing regimen. The results suggest that features are similar however at milder levels after intermittent dosing treatment than those observed in the mice that received the continuous dosing regimen. (A) Histopathology of the liver after continuous and intermittent therapy (LM). The images show areas of relatively normal liver tissue with no mass necrosis under hematoxylin-eosin staining. Scale bars=100 μm. (B) Glomeruli and tubular interstitia in xenograft and normal mice after intermittent therapy (TEM). Scale bars=2 μm.

Analysis of the renal tissues and cells revealed a similar trend. Mild glomerular inflammation and interstitial lesions were observed in the renal tissues of all of the xenograft mice under LM, and the changes were mild in the Chol-let-7a-treated group. However, significant congestion was observed in the glomeruli and tubular interstitia in the Chol-miRCtrl-treated group and the blank xenograft group (Fig. 5B). TEM analysis showed significant deposition of blood cells in the dilated glomerular capillaries in the Chol-miRCtrl-treated mice and blank xenograft mice (Fig. 5B). Mild membrane hyperplasia of the glomerular capillary walls and the presence of inclusion bodies in the glomerular capillary endothelial cells were observed in the Chol-let-7a- and Chol-miRCtrl-treated xenograft mice (4/4, 3/4). Mild dilation of the capillary lumens was seen in the Chol-let-7a- and Chol-miRCtrl-treated normal mice (3/4, 2/4) (Fig. 5B). Occasional irregular organelles were observed in the proximal tubular cells (data not shown).

Let-7a distribution and abundance in xenograft tumors and hepatic tissues after Chol-let-7a treatment Whole-animal imaging and frozen section analysis

Whole-animal imaging of nude mice and xenograft mice was performed to observe the distribution of Chol-let-7a and Chol-miRCtrl in vivo, and the overall fluorescence levels of Cy5-labeled Chol-let-7a and Chol-miRCtrl in the liver and kidney at different time points were analyzed using Indigo software. The whole-animal images showed high concentrations of fluorescence predominantly distributed in the liver, kidney, and bladder from 15 to 100 minutes post-injection (Fig. 6A and B), after which they began to decrease. At 19 hours after administration, the fluorescence was nearly undetectable in the liver, and fluorescence levels in the kidney and bladder region began to decrease.

F6
Figure 6.:
let-7a distribution and abundance levels in xenograft tumors and hepatic tissues after Chol-let-7a treatment. The distribution of Chol-let-7a and Chol-miRCtrl in vivo was examined by whole-animal imaging and frozen-sections under confocal and phase fluorescence microscopy observation. let-7a abundance in orthotopic tumors, corresponding paracancerous hepatic tissue, and normal liver tissue from healthy nude mice was semi quantitative detected by reverse transcription-polymerase chain reaction (RT-PCR). Whole-animal imaging and frozen-sections observation suggest that the cholesterol-conjugated molecules target to xenograft tumor and liver tissues. RT-PCR confirmed the situation. Whole-animal imaging suggests that kidney function of the xenograft mice was affected; excretion of the cholesterol-conjugated molecules was delayed. (A) Cy5-labeled Chol-let-7a and Chol-miRCtrl distribution observed from belly (Ventral whole-animal images). (B) Cy5-labeled Chol-let-7a and Chol-miRCtrl distribution observed from back (Dorsal whole-animal images). The signal strength increases as the color shifts from purple to blue, green, yellow, and red. (C, D) Overall fluorescence levels in the liver (C) and kidney (D) of normal and xenograft animals. The representative average overall signal levels from all groups are shown (n=2 mice per cohort). Chol-let-7a-N: Chol-let-7a-treated normal mice; Chol-miRCtrl-N: Chol-miRCtrl-treated normal mice; Chol-let-7a-X: Chol-let-7a-treated xenograft mice; Chol-miRCtrl-X: Chol-miRCtrl-treated xenograft mice. (E) Chol-let-7a and Chol-miRCtrl distribution in orthotopic tumor tissue and liver tissue. Sections taken 2 hours after injection with Chol-let-7a or Chol-miRCtrl were observed by confocal and phase fluorescence microscopy. The fluorescent confocal micrographs show Chol-let-7a or Chol-miRCtrl in the orthotopic tumor tissue and tumor-adjacent liver tissue. Original magnification: 40×. (F) Chol-let-7a and Chol-miRCtrl distribution in orthotopic tumor and other organ tissue in normal and xenograft mice. Fluorescence was observed 2 hours after injection with Chol-let-7a or Chol-miRCtrl. 0: tumor; 1: liver; 2: kidney; 3: heart; 4: lung; 5: spleen; 6: pancreas. The signal strength increases as the color shifts from purple to blue, green, yellow, and red. (G) Let-7a abundance in xenograft tumor tissue and adjacent liver tissue as determined by real-time PCR. Using miRNA-specific primers, relative let-7a levels were calculated using the comparative cycle threshold (CT) method (2−ΔΔCT method), with let-7a expression normalized to U6. The results shown represent the mean and standard error from three independent experiments. ** P<0.01 (Student's t-test).

Beginning at 19 hours after administration, differences in fluorescence levels and distribution among the groups became apparent. The patterns of fluorescence levels in the liver, kidney, and bladder showed similar trends in normal and xenograft mice injected with Chol-let-7a and Chol-miRCtrl. However, the concentrations of the fluorescently labeled Chol-let-7a and Chol-miRCtrl in the kidney and bladder decreased much more slowly in the xenograft mice. Moreover, the fluorescence patterns in the livers and kidneys of the xenograft mice differed over time between the Chol-let-7a and Chol-miRCtrl groups. The fluorescence signal was chiefly concentrated in the hepatorenal region from 15 to 100 minutes. However, the Chol-let-7a fluorescence concentration was much stronger than the Chol-miRCtrl signal, especially in the liver (Fig. 6C and D). As measured in fluorescence arbitrary units, Chol-let-7a fluorescence in the xenograft liver was 4.76 times that seen in Chol-miRCtrl xenograft mice, and 9.90 and 7.55 times that seen in Chol-let-7a-and Chol-miRCtrl-treated normal mice, respectively, at 100 minutes. At 19hours after administration, the fluorescence decreased rapidly, and was nearly undetectable at 48 hours in normal mice, but was still present at relatively high levels in the kidneys and bladders of both xenograft groups. The Chol-let-7a and Chol-miRCtrl fluorescence levels in the xenograft kidneys were 5.0938 and 4.0938 fluorescence arbitrary units, respectively, which corresponds to 4.77 and 4.08 times of those of the normal controls, respectively (Fig. 6D).

To further compare the fluorescence distribution in orthotopic tumors and adjacent hepatic tissue, frozen sections collected 2 hours post-injection of Chol-let-7a or Chol-miRCtrl were observed under confocal and phase fluorescence microscopy. In the xenograft mice, fluorescence was observed in both the orthotopic tumor tissues and hepatic tissues, and the intensity was stronger in the tumor tissue than in the corresponding normal hepatic tissue (Fig. 6E).

Next, fluorescence signals were observed in orthotopic tumor and other organ tissue samples taken 2 hours after injection of Chol-let-7a or Chol-miRCtrl into normal and xenograft mice, using confocal and phase fluorescence microscopy. Strong fluorescence signals were primarily observed in the xenograft tumors, livers, and kidneys from normal and xenograft mice; there was almost no fluorescence seen in other organs, such as the heart, lung, spleen, and pancreas (Fig. 6F). This was consistent with the results from the whole-animal imaging.

RT-PCR results

Using miRNA-specific primers and RT-PCR, we examined let-7a abundance in orthotopic tumor tissue, paracancerous hepatic tissue from xenograft mice after 5 weeks of Chol-let-7a, Chol-miRCtrl- and saline (blank) treatment, and heptic tissue from normal mice (Fig. 6G). The let-7a levels were significantly higher in Chol-let-7a-treated xenografts (Chol-let-7a vs Chol-miRCtrl, P = 0.003; Chol-let-7a vs blank xenograft, P = 0.001); they were 4.35 and 5.26 times those of the Chol-miRCtrl-treated and blank xenograft controls, respectively. There was no significant difference between the two xenograft control groups (Chol-miRCtrl vs Blank xenograft, P = 0.184). However, the let-7a levels in the orthotopic xenografts were still much lower than those seen in the paracancerous hepatic tissue (50.6%) and hepatic tissue in normal mice (40.7%). Moreover, let-7a was not significantly up-regulated in the paracancerous hepatic tissue compared to hepatic tissues from normal mice (P = 0.691).

Serum cytokines

In the xenograft mice, IL-6 (P< 0.05), TNF-α (P< 0.05), and VEGF (P < 0.05) levels were significantly lower in the Chol-let-7a-treated group than in the Chol-miRCtrl and blank xenograft groups (Additional Fig. 1AF1, https://links.lww.com/JR9/A31). Serum IL-8 levels were also decreased in the Chol-let-7a-treated group, but the differences were not significant (P > 0.05).

The levels of IL-6, IL-8, TNF-α, and VEGF in the Chol-let-7a-and Chol-miRCtrl-treated normal mice were not significantly different from those seen in the normal control mice (P > 0.05) (Additional Fig. 1BF1, https://links.lww.com/JR9/A31). Moreover, we found that IL-6, IL-8, TNF-α, and VEGF levels were significantly increased in the xenograft-only mice in comparison with the normal control mice (Additional Fig. 1CF1, https://links.lww.com/JR9/A31) (IL-6, P = 3.885 × 10-5; IL-8, P = 0.0076; TNF-a, P = 3.72 × 10-4; VEGF, P = 0.0467).

Discussion

Antitumor efficacy of Chol-let-7a delivered continuously or intermittently

Previous studies have primarily focused on identifying an effective delivery system for miRNA-based anticancer therapies, which are considered a major obstacle to their clinical use.[11] In clinical practice, a variety of anti-tumor drugs are usually used in combined regimens to decrease the dosage of each medicine used, thereby reducing their untoward effects and avoiding drug resistance. In addition, some drugs are administered intermittently to facilitate tissue and cell repair between doses. Based on this idea, we designed an intermittent dosing regimen for miRNA administration. Our results suggest that both continuous and intermittent Chol-let-7a dosing regimens inhibit tumor growth, although the continuous dosing regimen exhibited a greater capacity to shrink the tumors and resulted in fewer intrahepatic and distal (spleen and colon) metastases.

Potential characteristic morphological features of Chol-let-7a off-target effects on the liver and kidney

Safety is an important aspect of drug delivery systems intended for clinic use. The liver and the kidney often show signs of toxicity and injury as a result of exposure to drugs, xenobiotics, or chemicals.[18] Thus, understanding the features and potential mechanism of hepatotoxicity and nephrotoxicity of novel therapeutics is critical for pharmaceutical drug development. Consistent with this, whole-animal imaging showed that Chol-let-7a and Chol-miRCtrl primarily accumulated in the liver and kidney. Thus, we analyzed these organs in detail to evaluate the potential off-target effects of Chol-let-7a.

Histopathology is recognized as the single most appropriate screen for evidence of liver and kidney injury.[14] Cell injury can be noted by electron microscopy examination. Thus, the features of liver and kidney tissues and cells after treatment with the cholesterol-conjugated molecules were observed under LM and TEM to identify the effects of xenograft tumor growth, the delivery system, and up-regulated let-7a expression. Features that were only observed in Chol-let-7a- and Chol-miRCtrl-treated mice, and not found in blank xenograft mice, were considered to represent damage induced by Chol-let-7a therapy. Contrary, some histopathological and ultrapathological features that consistent with the growth of xenograft, especially found in blank xenograft mice, were not off-targets of the cholesterol-conjugated molecules,

We summarized and compared the features among the xenograft groups, as well as the differences between the xenograft and normal groups. These results in this study showed that the diffuse simusoidal dilation and edema, and the sinudoidal endothelial cell hyperplasis were related to Chol-let-7a and Chol-miRCtrl treatment. This was confirmed in the Chol-let-7a- and Chol-miRCtrl-treated normal mice. The characteristic changes in normal hepatic tissues and cells after administration of the cholesterol-conjugated molecules; however not found in blank xenograft mice, were diffuse sinusoidal dilatation with or without structural damage and sinusoidal lining cell hyperplasia. Similarly, dilation of the glomerular lumen and endocapillary membrane hyperplasia or edematous glomerular capillary and tubular interstitium, often accompanied by inclusion bodies in the glomerular endothelial cells, were primarily induced by injection of the cholesterol-conjugated molecules, Chol-let-7a and Chol-miRCtrl.

However, all of these features, which were found after Chol-let-7a treatment, are non-specific changes that are also produced by other drugs and treatments,[18] including the relative characteristic feature, diffuse sinusoidal dilatation in hepatic tissues, has also been reported in an oral contraceptive-induced liver injury.[19] Similarly, characteristic changes in the renal tissues and cells have also been reported as drug side effect. In addition, the liver and kidney effects were less severe in the intermittent dosing regimen group compared with the continuous regimen.

All these features found in xenograft mice treated with Chol-let-7a or Chol-miRCtrl exhibited tissue and cell features that are consistent with some common necroinflammatory patterns of drug-induced liver injuries, such as acute (lobular) hepatitis, chronic (portal) hepatitis, and zonal coagulative necrosis, were not characteristic features resulting from Chol-let-7a or Chol-miRCtrl. Some of the changes were accompanied by spotty and focal parenchyma cell degeneration, apoptosis, or necrosis. Furthermore, these features were aggravated in the corresponding Chol-miRCtrl-treated xenograft groups. Congestion and severe acute hepatitis with mass necrosis were mainly accompanied by xenograft tumor growth. In addition, congestion and inflammation were less severe in the Chol-let7a-treated xenograft group. To confirm this hypothesis, we examined serum cytokines from xenograft and healthy nude mice to analyze the potential effects of xenograft growth and Chol-let-7a on the immune state of nude mice, and to clarify the off-targets from some of the immuno-related changes of Chol-let-7a in xenograft mice. Serum cytokine changes in xenograft mice and normal mice after Chol-let-7a and Chol-miRCtrl suggested that the immune status of the xenograft nude mice affected mostly from the tumor growth. The levels of four cytokines IL-6, IL-8, TNF-α, and VEGF in the Chol-let-7a- and Chol-miRCtrl-treated normal mice were not significantly different from those seen in the normal control mice (P > 0.05). However, the serum levels of IL-6, IL-8, TNF-a, and VEGF in the blank xenograft group were significantly increased in the blank xenograft mice in comparison with the normal control mice.

Analysis of potential off-target effects of Chol-let-7a to liver

MiRNAs target multiple molecules, which could cause side effects at the molecular level in vivo. In this study, we analyzed the risks of up-regulating let-7a expression in mouse livers. First, we confirmed that let-7a was effectively delivered to the xenografted tumor and liver tissues, through in vivo whole-animal imaging and in vitro frozen section analysis. Under confocal and phase fluorescence microscopy, Chol-let-7a fluorescence was observed in both the orthotopic tumor tissues and adjacent liver tissues. The Chol-let-7a fluorescence signal was stronger than the Chol-miRCtrl fluorescence signal in the orthotopic tumor tissues. We then used RT-PCR to semiquantitatively detect let-7a levels in the tumor and adjacent hepatic tissues, and found that let-7a was significantly up-regulated in the tumor tissues but only slightly up-regulated in the paracancerous hepatic tissues. These results suggest that there is no major risk of let-7a up-regulation in healthy liver tissue having off-target effects.

Potential explanation for the delayed urinary excretion of cholesterol-conjugated molecules seen in the xenograft mice

The specific pattern of kidney toxicity and injury of a drug, xenobiotic, or chemical is dependent on many factors, such as its physiochemical properties (eg, low intrinsic solubility), dose, toxicokinetic properties, renal clearance profile, metabolic attributes, and length of exposure.[20] In this study, we were unable to determine the mechanism of the damage induced by the conjugates. We assessed morphological and ultrastructural tissue changes, as well as conjugate distribution and excretion, to determine why excretion of the cholesterol-conjugated molecules was delayed. There were no obvious differences in the Chol-let-7a and Chol-miRCtrl fluorescence in the liver and kidney of normal mice. However, in the xenograft mice, especially the Chol-miRCtrl and blank xenograft control groups, the fluorescence signals in the kidney decreased slowly and were still partially present at 72 hours post-injection. We believe that the delayed excretion was due primarily to kidney injury and inflammation, which resulted from orthotopic tumor growth.

A previous study reported that kidney parenchyma is especially vulnerable to the pro-inflammatory response to infection, and thus tissue damage, because it receives approximately 25% of the cardiac output.[14] Serum cytokine levels in this study confirmed this to some extent. In normal mice, cytokine levels were not altered by Chol-let-7a or Chol-miRCtrl treatment. The abundance of four cytokines was significantly increased in the xenograft mice compared with the normal control mice. However, the cytokine levels were significantly reduced after Chol-let-7a treatment. In addition, hepatic inflammation and necrosis were also decreased in the Chol-let-7a-treated xenograft mice. Thus, the cytokine levels seemed to be consistent with tumor size, degree of inflammation, and necrosis in the three xenograft groups. These results further suggest that the up-regulation of cytokines and inflammation were primarily associated with xenograft growth, rather than Chol-let-7a. Thus, the inhibition of kidney-mediated excretion seen in the xenograft mice most resulted from tumor growth, not toxicity of the cholesterol-conjugated molecules. Moreover, the results showed that Chol-let-7a treatment could alleviate renal injury by down-regulated cytokines indirectly through inhibiting tumor growth.

Limitations

In this study, the modification with cholesterol-conjugated molecules may function as miRNA, as well as ds-siRNA that could not be divided completely. We only analyzed and discussed based on miRNA functions.

Fluorescence patterns were only analyzed beginning at 100 minutes post-injection and continuing until 19 hours postinjection, owing to experimental limitations. Although this time period allowed us to roughly estimate the in vivo distribution and metabolism of these molecules, as well as observe the delay in urinary excretion, it did not enable us to track the complete roadmap of these molecules in the tumor and hepatic tissues.

Owing to the limitations of the experimental conditions, serum cytokines were examined using methods and devices designed for clinical use. In addition, the samples from each group were pooled for testing; thus, the results may not reflect the actual situation.

Conclusions

Both continuous and intermittent dosing with Chol-let-7a resulted in successful delivery of the conjugate to xenograft tumor tissues, as well as up-regulation of let-7a levels, and significantly inhibited tumor growth and metastasis.

Chol-let-7a treatment induced some non-specific liver and kidney damage, including diffuse sinusoidal dilatation with or without structural damage and sinusoidal lining cell hyperplasia in livers, and mild membrane hyperplasia in dilated or edematous glomerular capillaries and the tubular interstitium, often accompanied by the presence of inclusion bodies in the glomerular endothelial cells.

Chol-let-7a did not seem to have any severe off-target effects. In addition, it alleviates the inflammation and congestion caused by the tumor itself by inhibiting tumor growth.

Using an intermittent dosing regimen to reduce the damage associated with Chol-let-7a while maintaining satisfactory antitumor efficacy could be a useful strategy in future studies, and potentially clinical use.

Acknowledgments

The authors thank Mrs. Huimin Zhao, Wenyu Hao, and Huanxian Cui from the Centre for Experimental Animal Research (CEAR), Institute of Basic Medical Sciences, CAMS/ PUMC, as well as Xiao Yang (VisualSonics, Inc. Beijing, China) and Yi Gao (Berthold, Beijing, China) for the technical support. We also thank Dr. Wei-Min Tong for his constructive suggestions in support of this study. The authors thank Dr. Xingyi Hang and Ms. Jiahui Liu for their assistance with statistical analysis.

Author contributions

JG initiated the project. JG and ML analyzed data, wrote the manuscript, and performed most of the experiments. YX performed ultrasonic analysis. XL, WD, and YH performed electronic microscopic analysis. LZ, XD, TY, SG, XL, and YL performed molecular imaging, western blot, immunohistochemi-cal staining, hematoxylin-eosin staining, and analysis. All authors read and approved the final manuscript.

Financial support

This work was supported partly by the National Human and Health Scientific Data Sharing Platform, Clinical Center of China (No. 2004DKA20240-2014), and National Program funded by the Ministry of Science and Technology of China (No. 2015KJRK1L01). The content is solely the responsibility of the authors and does not represent the official views of the funding agencies.

Institutional review board statement

The experiments were approved by the Institutional Research Board of Peking Union Medical College Hospital.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Editor note: JG is an Editorial Board member of Journal of Bio-X Research. She was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer review handled independently of this Editorial Board member and their research groups.

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Keywords:

drug-induced renal injury; hepatic toxicity; in vivo off target effects; let-7 mimics; nonviral delivery vector

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