Abstract
Background/Aim: SN-38, an active metabolite of irinotecan, exhibits toxicity to all proliferating cells, causing dose-limiting and potentially life-threatening side effects. Newly established water-soluble derivatives of SN-38, 7-ethyl-9-(N-morpholinyl)methyl-10-hydroxycamptothecin (BN-MOA) and 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (BN-NMe), exhibit a unique mechanism of spontaneous alkylation of aromatic bases in DNA and show greater in vitro activity on cancer cell lines than SN-38. The aim of this study was to compare the therapeutic responses to irinotecan, BN-MOA and BN-NMe in vivo and in vitro in 3D cultures using colorectal cancer (CRC) patient derived xenografts (PDX). Materials and Methods: Seven established PDX tissues were subcutaneously grown on the flanks of NSG or NSG-SGM3 mice and tumor diameters were measured with a caliper. Compounds were administrated intraperitoneally at 40 mg/kg every five days. 3D PDX cultures were performed on 96-well LifeGel plates and cell viability was determined with the CellTiter Glo 3D reagent. Results: Treatment with irinotecan significantly delayed or stopped the growth of 5 out of 7 PDXs, with a greater level of inhibition from BN-MOA compared to irinotecan and BN-NMe. In vitro studies exhibited the same trends in SN-38 and BN-NMe but not in BN-MOA. Conclusion: The new SN-38 derivatives, BN-MOA and BN-NMe, showed enhanced therapeutic effects compared to irinotecan in CRC models. BN-MOA demonstrated superior tumor inhibition in vivo, while BN-NMe had similar in vitro activity to SN-38. These findings highlight the potential of BN-MOA for greater antitumor efficacy in vivo, with BN-NMe showing comparable effectiveness to SN-38 in vitro. Future studies should optimize growth models to better predict anticancer drug responses.
Colorectal cancer (CRC) is the third most common cancer worldwide. In 2018, 1.8 million new cases of CRC were diagnosed and 881,000 deaths were reported (1, 2). Although CRC mortality can be reduced by cancer prevention and early detection, once a tumor has metastasized to secondary locations, the chances of curative treatment drop significantly. The chemotherapeutic treatment of CRC is based mainly on cytotoxic drugs, including the combination of 5-fluorouracil with leucovorin and oxaliplatin (FOLFOX) and the combination of 5-fluorouracil with leucovorin and irinotecan (FOLFIRI) (3, 4). In advanced CRC patients, cytotoxic drugs are often used in combination with or replaced by targeted therapies, including monoclonal antibodies for vascular-endothelial growth factor or epidermal growth factor receptor, BRAF inhibitors, and immune checkpoint inhibitors (5). Cytotoxic drugs are highly effective in in vitro assays but exhibit only modest selectivity between malignant and normal cells, resulting in numerous adverse side effects that often prevent their full therapeutic effect in cancer patients. In turn, targeted therapies against precisely defined molecular targets of carcinogenesis, such as constitutive activation of cell proliferation or inhibition of apoptosis, can achieve a higher therapeutic index only in some specific cancer phenotypes.
Irinotecan (also known as CPT-11), a water-soluble precursor drug, which is a semi-synthetic analogue of the plant alkaloid camptothecin, acts as a topoisomerase I (Topo I) inhibitor and is one of the most important antineoplastic drugs used for the treatment of metastatic CRC and other solid tumors (6). Irinotecan is converted to its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), by carboxylesterase 2 in blood and liver. Most SN-38 is excreted in the bile as inactive SN-38-glucuronide conjugates, which, in turn, can be deconjugated with β-glucuronidase from intestinal microflora. SN-38 exhibits toxicity to all proliferating cells, causing dose-limiting and potentially life-threatening side effects, such as delayed onset diarrhea, myelosuppression and hepatotoxicity, in a significant proportion of cancer-treated patients (7-9). Newly established water-soluble derivatives of SN-38, including 7-ethyl-9-(N-morpholinyl)metyl-10-hydroxycamptothecin (BN-MOA) and 7-ethyl-9--(N-methylamino)methyl-10-hydroxycamptothecin (BN-NMe) [Patent No: EP2912039B1 and (10) US 9,682,992 B2 (11)], exhibit a unique mechanism of spontaneous alkylation of aromatic bases in DNA and act as the most effective blocks of Topo I activity (12). As a result, they show several orders of magnitude greater in vitro activity in breast, leukemia, colon, and lung cancer cell lines than irinotecan clinically used as an API of Pfizer’s CAMPTO formulation (11). Since these derivatives have limited metabolism to SN38, the side effects of this compound may also be limited. However, further preclinical studies are required before these camptothecin analogs are sent for clinical studies.
In this study, we compared the therapeutic responses to irinotecan and two derivatives of SN-38, BN-MOA and BN-NMe, analyzed using xenografts derived from CRC patients in in vivo studies and their growth of 3D cultures in vitro. We found comparable antitumor activity of irinotecan and the analogues of the SN-38 derivatives when tested in vivo. However, in vitro studies exhibited the same trends in SN-38 and BN-NMe but not for BN-MOA activity.
Materials and Methods
Mouse breeding. NSG (NOD scid gamma) mice and NSG-SGM3 (NSGS) mice, purchased from The Jackson Laboratory (Bar Harbor, ME, USA), were maintained in a specific pathogen-free facility (SPF) under proper environmental conditions (temperature, humidity, and 12 h light cycle) with free access to water and food. PDX experiments. This study used 7 different CRC patient-derived xenografts (PDXs), previously established at Maria Sklodowska-Curie National Institute of Oncology and grown in NU/J mice. Pieces of PDXs (~10-20 mm3) freshly resected from NU/J mice (generation P3-P7) were cryopreserved in a medium containing 50% DMEM, 40% FBS, and 10% DMSO. After thawing, pieces of tumors were subcutaneously implated on both flanks of 2-4- or 6-10-week-old NSG or NSG-SGM3 mice, respectively. When the tumor volume reached 1,000-1,500 mm3, tissue was resected and implanted on the left flank of experimental sets of mice (6-10-week-old, both sexes NSG or NSG-SGM3 mice). Animals were assigned randomly to treated and control groups when tumors exceeded a volume of 150 mm3. All experimental procedures performed on mice were performed in accordance with the EU Directive 2010/63/EU and approved by the 2nd Local Ethics Committee for Animal Experimentation in Warsaw (WAW2/014/2020 from 29th January 2020).
Administration of chemotherapeutic drugs. The groups consisted of 6-8 mice. Irinotecan and its two newly established derivatives, BN-NMe and BN-MOA (12) were administered intraperitoneally (IP) at a dose of 40 mg/kg each at five-day intervals, and the control groups received 0.9% saline vehicle treatments. The mice were carefully observed for signs of distress. Mice were weighed, the tumor size was measured with a caliper before drug administration, and tumor volume was calculated using the following formula: (Length × Width × Width)/2, where W is the shortest diameter and L is the longest diameter (13). Mice within a set were sacrificed when the tumor size of most tumors in the control groups reached approximately 1,200 mm3 or earlier when symptoms of distress appeared, and necropsy was performed to collect blood and tissues for further analysis. Animals were euthanized in 3 timepoints: 15, 30 and 60 min after the last drug administration.
Cell culture media for PDX culture and transportation. PDX culture medium (PDX-KOKS, R171611, Real Research, Krakow, Poland) was comprised of 10% (v/v) fetal bovine serum (FBS) (EURx Ltd, Gdansk, Poland), 34% (v/v) DMEM-F12 (1:1) (Gibco, Waltham, MA, USA), 31% (v/v) L-WRN (ATCC, CRL-3276) conditioned medium which was collected according to the manufacturer’s protocol, 1x GlutaMax (Gibco), 20 mM HEPES buffer (Gibco), 2% B27RR Supplement (B27RR, R161611, optimized by Real Research), 2% N2RR Supplement (N2RR, R151611, optimized by Real Research), 1x antibiotic cocktail (BactiSafe-RR, R131211, Real Research) and an optimized combination of factors described previously in the literature (14-16). The transportation medium consisted of DMEM-F12 (1:1) supplemented with 20% FBS, 1% (v/v) antibiotic cocktail and 10 μM Y-27632 (TargetMol, Boston, MA, USA).
Cell preparation from PDX tissue. Freshly resected PDXs, used for in vivo studies, were placed in a transportation medium and sent to Real Research headquarters in a transportation box at 4°C. After approximately 24-48 h, tissue fragmentation and digestion were performed. PDX tissue was added to a 5 ml PBS supplement with 1x antibiotic cocktail in a petri dish and manually cut into small fragments using surgical scissors. The suspension of PDX fragments was removed into a preweighed tube, the petri dish was rinsed with an additional portion of PBS with the antibiotic cocktail, and the pooled mixture was centrifuged at 400 × g for 10 min. After supernatant aspiration, the remaining weight of the PDX material was determined before resuspending in a tissue digestion kit (TDK-RR, R111113, Real Research) at 1 ml/g of tissue. Incubation at 37°C for 60-90 min (until the majority of large fragments were digested) was interspersed with brief mixing every 10 min. Two volumes of DMEM-F12 medium supplemented with 20% FBS and 1x antibiotic cocktail were added to the digestion mix before centrifugation at 400 × g for 10 min. The supernatant was removed, and the digested cell pellet was resuspended in 5 ml of DMEM-F12/20% FBS/1x antibiotic cocktail, before passing through a prewetted 70 μm cell strainer (Falcon Corning, Corning, NY, USA). The strained cell filtrate was re-pelleted by centrifugation. Some contaminating erythrocytes were evident by visual inspection of the pellet, so the pellet was resuspended in 1x RBC lysis solution and processed according to the manufacturer’s instructions (BioLegend, San Diego, CA, USA). Finally, the clarified cell pellet was resuspended in a prewarmed PDX-KOKS culture medium followed by microscopic live cell counting based on trypan blue dye exclusion.
3D cell growth and drug sensitivity testing. 96-well LifeGel plates for PDX 3D cell culture supplied in DMEM-F12 medium (Real Research, L915131212) were pre-equilibrated at 37°C and 5% CO2. The medium was removed above the LifeGel layer before seeding with 10,000 cells in 150 μl of PDX culture medium per well, after which the plates were returned to the humidified incubator (Day 0). On day 2, 120 μl of culture medium was removed from the wells and replaced with the same volume of fresh medium. The culture medium was removed again, just before the start of drug treatment on day 5.
A total of 10 mM solutions of SN-38, the active metabolite of irinotecan and its analogues, were made in DMSO and 3-fold drug dilution series were prepared in PDX culture medium, maintaining the same concentration of DMSO throughout. 75 μl of each drug dilution or control medium were added to the wells. The drug concentration data reflects the final concentration after diffusion into a total of 200 μl, and the final concentration of DMSO in wells was 0.1 % (v/v) throughout. Cells were exposed to drugs for 3 days before performing viability assays.
Cell viability was determined by quantitating intracellular ATP concentrations using the CellTiter Glo 3D reagent according to the manufacturer’s guidelines (Promega, Madison, WI, USA). Culture plates were cooled to room temperature for more than 45 min before adding 75 μl of reagent and leaving them for another 30 min at room temperature. Bioluminescence counts were quantitated using a Victor2 plate reader (Perkin Elmer, Waltham, MA, USA).
Technical triplicate dose-response curves were performed for each PDX sample except when otherwise stated, and each of the three response curves consisted of duplicate data points. The bioluminescence values were corrected for background by subtracting no-cell control values; they were then normalized for comparison between the PDX samples by setting no-drug control values to 100%. The IC50 calculations were performed using http://www.grcalculator.org/.
Statistical analysis. Differences between groups were evaluated using the two-way repeated measures ANOVA test. A p<0.05 was considered significant. The analyses were performed using GraphPad v8.2 (GraphPad Software, San Diego, CA, USA).
Results
In vivo studies. The potential of BN-NMe and BN-MOA as therapeutic agents was compared to the potential of irinotecan using seven previously established CRC PDX models (17). The body weight of the mice was measured at 5-day intervals to an accuracy of 0.1g. No significant differences were observed in the body weights of mice in the control and drug injection groups (data not shown). In control groups that received 0.9% saline vehicle treatments, a high degree of inter-individual variability of the growth of 4 PDXs (X39, X41, X49 and X154) was observed; the duration of the experiment between the first drug injection and the end of observation ranged between 15 and 30 days. Treatment with irinotecan significantly delayed or stopped the growth of all PDXs, except for two (X49 and X154), as shown in Figure 1. Of the PDXs that responded to irinotecan, X35 and X49 grew the fastest, while X41, X52 and X154 grew the slowest and showed the highest spread of tumor size. BN-NMe treatment of mice with tumors significantly decreased the growth of five PDXs, except for two PDXs, X35 and X154. In turn, BN-MOA showed growth inhibition in all six PDXs tested, with a level of inhibition greater than that revealed by irinotecan and BN-NMe (Figure 1).
In vivo studies show the decreased growth of patient-derived xenografts (PDXs) after treatment with irinotecan and two analogues of SN-38, BN-NMe and BN-MOA, evaluated as fold change between the tumor volume before drug administration and the volume at the end of the experiment. Drugs were administered intraperitoneally at a dose of 40 mg/kg each at 5-day intervals. Group size n=5-8, each mouse carried one tumor; error bars show standard error of the mean (SEM). Differences between groups were evaluated using two-way ANOVA test followed by Dunnett’s correction; ns, not significant; *p<0.05 and **p<0.01.
In vitro studies. Next, the cytotoxic effect of SN-38 and its analogues, BN-NMe and BN-MOA, was compared using in vitro studies. Cells isolated from freshly resected PDX and matching tissues used for in vivo studies, were grown for 5 days as 3D cell cultures and then exposed to a 3-fold drug dilution series followed by cell viability determination by quantitating intracellular ATP concentrations. PDX X29 and X35 were the most sensitive to compound SN-38 with an IC50 equal to 0.0034 μM and 0.058 μM, respectively. All other PDX were moderately sensitive with IC50 concentrations between 0.18 and 0.53 μM. Although the cytotoxic activity of the BN-NMe compound was found to be similar to the activity of SN-38, the BN-MOA compound showed a 10-fold lower cytotoxic activity in each culture (Table I, Figure 2).
Cytotoxic potency (as measured by the half maximal inhibitory concentration, IC50) of BN-NMe, BN-MOA and SN38 tested in 3D cell cultures from seven colorectal cancer patient-derived xenografts (PDXs).
IC50 plots for the PDX models grown as 3D cultures. The dose-dependent cytotoxicity of SN-38, BN-NMe, and BN-MOA against 3D cultures from the PDX cell preparation was calculated from the inhibition of control cell viability measurements based on cellular ATP levels. IC50 determinations were performed in triplicate in each single biological sample for each compound and IC50 values were derived from duplicate test points for all nine concentrations of compounds and controls. Graphs show the mean±standard error of the mean (SEM). X-axis=log10 [IC50 (μM)].
Discussion
Herein we compared the therapeutic responses to irinotecan and the two new water-soluble derivatives of SN-38, BN-NMeR and BN-MOA that were tested in vivo using CRC PDX models and in vitro using the matching 3D cultures. While in vivo studies showed comparable antitumor activity between irinotecan and the two derivatives of SN-38, in vitro studies showed a similar response to SN-38 and BN-NMe, but not to the BN-MOA compound.
PDX models have been used for over half of a century in cancer research for their ability to closely mimic the histopathology, genetic characteristics, and drug response of the original patient tumors, making them invaluable for preclinical trials, drug development, and personalized oncology (17). A decade ago, patient-derived organoids (PDO) were used for the first time in cancer research (18, 19) and are regarded as a breakthrough technology with enormous potential for cancer biology and personalized therapy. 3D cultures utilizing patient cells have advantages including operational convenience and cost and can be expanded rapidly to allow high-throughput genes and drug screenings when compared to PDX models. We do not categorize the 3D culture method used in the current work as organoids since our approach does not rely on ensuring that the 3D culture mimics the in vivo physiology and structure of an organ, the features that are inherent to an organoid (20). Instead, the model used in our work would fall into a definition of a tumoroid where cancerous cells grow centrically into multiple layers forming the dimensionality of cell-cell interactions and ultimately generating an in vitro 3D model (21).
Both PDXs and a 3D cultures utilizing either PDOs or PDX-derived organoids (PDXO) have been used in the past to test responses to irinotecan. For example, a study by Ooft et al. revealed that PDO models predict response to irinotecan monotherapy in metastatic CRC patients (22). A recent work by Guillen et al. reported the development of matched PDX and PDXO cultures, particularly focusing on treatment-resistant and metastatic breast cancers. They found that PDXOs could accurately predict drug responses seen in PDXs, including for irinotecan (23). In our work in vivo and in vitro models showed significant differences in drug sensitivity, depending on the PDX model used. Two PDXs, X29 and X35, with the highest growth rate in mice also showed the highest therapeutic responses to the three compounds studied. 3D cultures of X29 and particularly X35 also grew significantly faster than all other samples tested, and their faster growth rates resulted in the lowest IC50 values. Therefore, the antineoplastic activity of the derivatives of irinotecan and SN-38 in in vivo and in vitro studies may be related to tumor growth rates and 3D cultures. However, although the measurement of the tumor growth rate in an organism is evident, it is still very challenging to determine accurate growth rates for 3D structures. Although a preferred cross-culture technology comparison for inhibitors is based on inhibition of growth rates, slow-growing cultures of relatively quiescent cells may not progress to replicating 3D structures in vitro. Due to the spatial configuration of in vitro 3D systems, which can recapitulate tumor complexity, 3D models are promising for high-throughput screening of existing and novel therapies in preclinical and clinical settings (24). Ultimately, PDXs and 3D cultures are currently complementary approaches in cancer research allowing experimental flexibility and a more comprehensive understanding of tumor biology and drug responses, potentially improving the success rate of drug development and personalized treatment strategies (25).
Conclusion
While the development of therapeutics for anticancer therapies is a relatively slow process, new anticancer drugs are mostly identified by high-throughput technologies in studies conducted ex vivo and in vivo. A consequence of the use of inadequate and simplified preclinical models is that only 5-10% of the anticancer agents that passed preclinical testing were approved for use by the FDA (26, 27). Our studies also confirm the need for careful selection of the methods used in preclinical studies. The most significant differences between our in vivo and in vitro studies resulted from the treatment time. PDX models allowed 2 to 5 repeated drug administrations depending on their growth rates, while in 3D models the effect of a single drug administration to cultures was measured after the standard time of 5 days. To develop predictive models for new anticancer drugs, the defined growth rates of both PDX and 3D cultures should also be taken into account to achieve the desired results in future studies.
Footnotes
Authors’ Contributions
Conceptualization, JO, MM, LK, MK; methodology, KUB, MCL, DE, EK, ZSM, IR, MD, MS, BN, MU, JKK, KK and MK; formal analysis, JO, MM, DE and MK; investigation, KUB, MCL, DE, EK, ZSM, IR, MD, MS, BN, MU, JKK, KK and MK; writing – original draft, JO and MM; writing – review and editing, JO, MM, MK and DE; visualization, JO and MM; supervision, JO and MK; project administration, JO and MM; funding acquisition, JO. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
Marcin Krzykawski is the co-founder and CEO of Real Research. David Earnshaw, Justyna Kocik-Krol and Krzysztof Klimkiewicz are employees of Real Research.
Funding
This research was funded by Polish National Science Center, grant number 2018/31/B/NZ7/02675 to JO.
- Received May 31, 2024.
- Revision received July 13, 2024.
- Accepted August 16, 2024.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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