Figure 1 - available via license: Creative Commons Attribution 3.0 Unported
Content may be subject to copyright.
Tumorigenesis in orthotopic and subcutaneous models was distinct. Orthotopic tumors were established by endoscopy-guide microinjection in the colon wall of BABL/c mice. In some mice, tumors and tumor-caused colon obstruction can be seen directly in the colon lumen (A, i-iii). In other mice, abnormal movement, stiffness, and bleeding can be seen in the colon (A, iv-vi). H&E staining showed tumor tissue in the submucosa layer. The invasion margin can be seen in the muscularis externa (A, vii). An in vivo imaging system can be used for monitoring in our orthotopic model (B). Injection of different numbers of CT26 cells (10 5 and 10 6 ) led to a significance difference in tumor volume at autopsy (C). At 4 weeks after injection of 10 3 HT29 tumor cells in either the colon wall or the subcutaneous connective tissue of athymic nude mice (n = 4 in each model), tumorigenesis occurred only in the subcutaneous model (D). Arrows indicate the representative injection sites in the colon wall, but no tumors formed (D, i-ii). At 4 weeks after injection of 10 4 CT26 cells in either the colon wall (E, i; n = 3) or the subcutaneous connective tissue (E, ii; n = 4) of BALB/c mice, subcutaneous tumors formed (E, i); however, no orthotopic tumors formed (E, ii). Autopsy confirmed the results of imaging (data not shown) (E). Injection of 10 5 MC38 did not induce tumor formation in the colon wall (n = 8) of C57/B6 mice, whereas it did cause subcutaneous tumor formation (n = 8) (F). H&E: hematoxylin and eosin. **P < 0.01.
Source publication
Existing preclinical models of human colorectal cancer (CRC) that rely on syngeneic subcutaneous grafts are problematic, because of increasing evidence that the immune microenvironment in subcutaneous tissue is significantly different from the gastrointestinal tract. Similarly, existing orthotopic models that use a laparotomy for establishing graft...
Contexts in source publication
Context 1
... we used endoscopy to monitor tumor growth, we saw abnormal protrusions in the colon lumen around 1 week after injection in some mice ( Figure 1A, i-ii); subsequently, rapid tumor growth induced colonic obstruction ( Figure 1A, iii). In other mice, as tumors invaded the submucosal layer and expanded toward the serosa, we could not detect tumor growth by bright-light endoscopy; however, we did see stiffness, brittleness, and heavy bleeding in the mucosa ( Figure 1A, iv-vi). ...
Context 2
... we used endoscopy to monitor tumor growth, we saw abnormal protrusions in the colon lumen around 1 week after injection in some mice ( Figure 1A, i-ii); subsequently, rapid tumor growth induced colonic obstruction ( Figure 1A, iii). In other mice, as tumors invaded the submucosal layer and expanded toward the serosa, we could not detect tumor growth by bright-light endoscopy; however, we did see stiffness, brittleness, and heavy bleeding in the mucosa ( Figure 1A, iv-vi). ...
Context 3
... we used endoscopy to monitor tumor growth, we saw abnormal protrusions in the colon lumen around 1 week after injection in some mice ( Figure 1A, i-ii); subsequently, rapid tumor growth induced colonic obstruction ( Figure 1A, iii). In other mice, as tumors invaded the submucosal layer and expanded toward the serosa, we could not detect tumor growth by bright-light endoscopy; however, we did see stiffness, brittleness, and heavy bleeding in the mucosa ( Figure 1A, iv-vi). Histologic analysis indicated that orthotopic tumors were growing in the submucosa layer and invading the muscularis layer ( Figure 1A, vii). ...
Context 4
... other mice, as tumors invaded the submucosal layer and expanded toward the serosa, we could not detect tumor growth by bright-light endoscopy; however, we did see stiffness, brittleness, and heavy bleeding in the mucosa ( Figure 1A, iv-vi). Histologic analysis indicated that orthotopic tumors were growing in the submucosa layer and invading the muscularis layer ( Figure 1A, vii). We detected orthotopic tumors around 3 weeks after implantation of 10 5 CT26- Luc cells ( Figure 1B) by IVIS. ...
Context 5
... analysis indicated that orthotopic tumors were growing in the submucosa layer and invading the muscularis layer ( Figure 1A, vii). We detected orthotopic tumors around 3 weeks after implantation of 10 5 CT26- Luc cells ( Figure 1B) by IVIS. A direct correlation between tumor volume and the number of injected tumor cells can be found in our orthotopic model ( Figure 1C). ...
Context 6
... detected orthotopic tumors around 3 weeks after implantation of 10 5 CT26- Luc cells ( Figure 1B) by IVIS. A direct correlation between tumor volume and the number of injected tumor cells can be found in our orthotopic model ( Figure 1C). ...
Context 7
... athymic nude mice, orthotopic injection of 10 3 HT29 cells could induce tumor formation at 4 weeks in the subcutaneous model, but not in orthotopic model ( Figure 1D). Orthotopic injection of 10 5 HT29 cells was sufficient to induce tumor formation and invasion at 4 weeks in the orthotopic model (Supplementary Figure 1A). ...
Context 8
... athymic nude mice, orthotopic injection of 10 3 HT29 cells could induce tumor formation at 4 weeks in the subcutaneous model, but not in orthotopic model ( Figure 1D). Orthotopic injection of 10 5 HT29 cells was sufficient to induce tumor formation and invasion at 4 weeks in the orthotopic model (Supplementary Figure 1A). Similar results were observed in our two syngeneic orthotopic CRC models. ...
Context 9
... results were observed in our two syngeneic orthotopic CRC models. For example, injection of 10 4 CT26 cells induced tumor formation at 4 weeks in subcutaneous tissue, but not in the colon wall in BALB/c mice ( Figure 1E; results confirmed by autopsy, data not shown) and injection of 10 5 MC38 cells induced tumor formation at 4 weeks in subcutaneous tissue, but not in the colon wall in C57BL/6 mice ( Figure 1F). Taken together, our data indicate that more cells are required to initiate tumorigenesis in the orthotopic model compared to subcutaneous model. ...
Context 10
... results were observed in our two syngeneic orthotopic CRC models. For example, injection of 10 4 CT26 cells induced tumor formation at 4 weeks in subcutaneous tissue, but not in the colon wall in BALB/c mice ( Figure 1E; results confirmed by autopsy, data not shown) and injection of 10 5 MC38 cells induced tumor formation at 4 weeks in subcutaneous tissue, but not in the colon wall in C57BL/6 mice ( Figure 1F). Taken together, our data indicate that more cells are required to initiate tumorigenesis in the orthotopic model compared to subcutaneous model. ...
Citations
... Accordingly, a recent study reported significant differences in the immune response when MC38 cells are injected subcutaneously or into the colon 52 , fueling the growing body of research suggesting that the tumor microenvironment differs considerably between organs 53,54 . Specifically, a higher level of tumor-infiltrating T cell and NK cells (around 3 times more) was measured in orthotopic CRC tumors compared to subcutaneous tumors 52 . As a consequence, orthotopic CRC showed a greater sensitivity to immune checkpoint blockade compared to subcutaneous tumors, demonstrating the important role of T cells in mediating tumor rejection within colon. ...
Development and progression of malignancies are accompanied and influenced by alterations in the surrounding immune microenvironment. Understanding the cellular and molecular interactions between immune cells and cancer cells has not only provided important fundamental insights into the disease, but has also led to the development of new immunotherapies. The C-type lectin Dendritic Cell ImmunoReceptor (DCIR) is primarily expressed by myeloid cells and is an important regulator of immune homeostasis, as demonstrated in various autoimmune, infectious and inflammatory contexts. Yet, the impact of DCIR on cancer development remains largely unknown. Analysis of available transcriptomic data of colorectal cancer (CRC) patients revealed that high DCIR gene expression is associated with improved patients’ survival, immunologically "hot" tumors and high immunologic constant of rejection, thus arguing for a protective and immunoregulatory role of DCIR in CRC. In line with these correlative data, we found that deficiency of DCIR1, the murine homologue of human DCIR, leads to the development of significantly larger tumors in an orthotopic murine model of CRC. This phenotype is accompanied by an altered phenotype of tumor-associated macrophages (TAMs) and a reduction in the percentage of activated effector CD4⁺ and CD8⁺ T cells in CRC tumors of DCIR1-deficient mice. Overall, our results show that DCIR promotes antitumor immunity in CRC, making it an attractive target for the future development of immunotherapies to fight the second deadliest cancer in the world.
... Для моделирования ксеногенных моделей различных онкологических патологий (РМЖ, рак пищевода, рак щитовидной железы и др.) используются несколько способов трансплантации стандартных опухолевых клеточных линий: подкожный, ортотопический [13]. С точки зрения особенности модели, способов моделирования и доклинических возможностей, каждый из перечисленных способов трансплантации опухолевых клеток имеет свои сильные и слабые стороны. ...
Важную роль в экспериментальных исследованиях противоопухолевых соединений играют модели ксеногенных опухолей in vivo. Несмотря на достигнутые в этой области успехи, вероятность того, что новое лекарственное противоопухолевое средство будет одобрено для использования в онкологической клинике, остается невысокой. Такая низкая воспроизводимость экспериментальных результатов в клинике связана с тем, что существующие стандартные модели злокачественных новообразований in vivo, имеют невысокую прогностическую значимость. Тем не менее эксперименты на лабораторных моделях ксеногенных опухолей играли и продолжают играть ключевую роль в апробации и продвижении потенциальных противоопухолевых препаратов в клинику. В данном обзоре представлен анализ отечественной и зарубежной литературы, посвященной созданию и применению ксеногенных моделей in vivo в области экспериментальной онкологии.
... Then, 2 × 10 5 CT26 cells were injected into the opposite flank as secondary tumors. In the CT26 model, a subcutaneous tumor is the less favorable situation for the immune response [21]. ...
Radiofrequency ablation (RFA) of cancer induces an anti-tumor immunity, which is insufficient to prevent recurrences. In mice, RFA–intratumoral immunotherapy by granulocyte–macrophage colony-stimulating factor (GM-CSF) and Bacillus Calmette-Guerin resulted in complete metastases regression. Infectious risk in human needs replacement of live vaccines. Intratumoral purified protein derivatives (PPD) have never been tested in digestive cancers, and the safety of intratumoral immunotherapy after RFA has not yet been validated in human models. We investigated the therapeutic efficacy of combined radiofrequency ablation (RFA) and intratumoral immunotherapy (ITI) using an immune-muco-adherent thermogel (IMT) in a mouse model of metastatic colorectal cancer (CRC) and the safety of this approach in a pig model. Intratumoral stability of the immunogel was assessed using magnetic resonance imaging (MRI) and bioluminescent imaging. Seventy-four CT26 tumor-bearing female BALB/c mice were treated with RFA either alone or in combination with intratumoral IMT. Regression of distant metastasis and survival were monitored for 60 days. Six pigs that received liver radiofrequency and intralesional IMT injections were followed for 15 days. Experimental gel embolisms were treated using an intravascular approach. Pertinent rheology of IMT was confirmed in tumors, by the signal stability during 3 days in MRI and 7 days in bioluminescence imaging. In mice, the abscopal effect of RFA–intratumoral immunotherapy resulted in regression of distant lesions completed at day 16 vs. a volume of 350 ± 99.3 mm3 in the RFA group at day 25 and a 10-fold survival rate at 60 days. In pigs, injection of immunogel in the liver RFA area was safe after volume adjustment without clinical, hematological, and liver biology disorder. Flow cytometry showed an early increase in CD3 TCR+T cells at D7 (p < 0.05) and a late decrease in CD29+-CD8 T cells at D15 (p < 0.05), reflecting the inflammation status changes. Systemic GM-CSF release was not detectable. Experimental caval and pulmonary thermogel embolisms were treated by percutaneous catheterism and cold serum infusion. RFA–intratumoral immunotherapy as efficient and safe mini-invasive interventional oncology is able to improve ablative treatment of colorectal liver metastases.
... Further, subcutaneous xenograft models don't result in metastasis due to less vascularization compared to the orthotopic model. Additionally, subcutaneous implantation results in less invasive potential, decreased antitumor immune response, and sensitivity to immune checkpoint blockade therapy [92]. Xenograft mouse models can also be used for developing CRC cell lines from transgenic mouse models with the mutations of CRC driving genes like APC, KRAS, and mutations in tumor suppressor TP53 and implanted in athymic nude mice and subsequently in the same genetic background animals to determine the tumorigenic potential. ...
... Studies concerning testing ICIs should carefully consider using an appropriate tumor model because tumor location has been reported to heavily influence immune responses in CRC animal models. The CRC orthotopic animal model very closely recapitulates the human CRC tumor microenvironment and presents comparable levels of tumor immune infiltration [39]. Therefore, we opted for the orthotopic model, a more clinically relevant model in which tumor cells were injected into the colonic mucosa using endoscopy. ...
... Although a significant difference in granzyme b production was noted between WT and KO tumors, no statistical difference was detected between the control and KO tumors. It is established that compared to orthotopic models, the subcutaneous model generally has fewer T and B cell infiltration [39]. ...
Colorectal cancer (CRC) currently ranks as the third most common cancer in the United States, and its incidence is on the rise, especially among younger individuals. Despite the remarkable success of immune checkpoint inhibitors (ICIs) in various cancers, most CRC patients fail to respond due to intrinsic resistance mechanisms. While microsatellite instability-high phenotypes serve as a reliable positive predictive biomarker for ICI treatment, the majority of CRC patients with microsatellite-stable (MSS) tumors remain ineligible for this therapeutic approach. In this study, we investigated the role of centrosomal protein 55 (CEP55) in shaping the tumor immune microenvironment in CRC. CEP55 is overexpressed in multiple cancer types and was shown to promote tumorigenesis by upregulating the PI3K/AKT pathway. Our data revealed that elevated CEP55 expression in CRC was associated with reduced T cell infiltration, contributing to immune exclusion. As CRC tumors progressed, CEP55 expression increased alongside sequential mutations in crucial driver genes (APC, KRAS, TP53, and SMAD4), indicating its involvement in tumor progression. CEP55 knockout significantly impaired tumor growth in vitro and in vivo, suggesting that CEP55 plays a crucial role in tumorigenesis. Furthermore, the CEP55 knockout increased CD8+ T cell infiltration and granzyme B production, indicating improved anti-tumor immunity. Additionally, we observed reduced regulatory T cell infiltration in CEP55 knockout tumors, suggesting diminished immune suppression. Most significantly, CEP55 knockout tumors demonstrated enhanced responsiveness to immune checkpoint inhibition in a clinically relevant orthotopic CRC model. Treatment with anti-PD1 significantly reduced tumor growth in CEP55 knockout tumors compared to control tumors, suggesting that inhibiting CEP55 could improve the efficacy of ICIs. Collectively, our study underscores the crucial role of CEP55 in driving immune exclusion and resistance to ICIs in CRC. Targeting CEP55 emerges as a promising therapeutic strategy to sensitize CRC to immune checkpoint inhibition, thereby improving survival outcomes for CRC patients.
... We recently showed that human soluble TRAIL (hsTRAIL), secreted intratumorally by non-pathogenic, Lactococcus lactic bacteria, was able to reduce the growth of subcutaneous HCT116-tumor in NOD-SCID mice and this effect could be further enhanced by oral administration of metformin (MetF) (Kaczmarek et al., 2021). However, data demonstrate that existing preclinical subcutaneous models of human colorectal cancer (CRC) markedly differ from the orthotopic transplants (Zhao et al., 2017), e.g. orthotopic CRC models follow the same metastatic patterns as in humans, while subcutaneous grafts are non-invasive (Zhang et al., 2013;Guilbaud et al., 2001). ...
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of sensitive cancer cells, including colorectal cancer (CRC). Due to its short biological half-life after intravenous administration and related clinical ineffectiveness, novel formulations of TRAIL need to be developed. Here we propose Lactococcus lactis bacteria as a vehicle for local delivery of human soluble TRAIL (hsTRAIL) in CRC. The use of common probiotics targeting guts as carriers for TRAIL could ensure its sustained release at the tumor site and extend the duration of its activity. We have already engineered hsTRAIL-secreting L.lactis bacteria and showed their effectiveness in elimination of human CRC cells in vitro and in vivo in a mouse subcutaneous model. Here, L.lactis (hsTRAIL+) were administered by gastric gavage to SCID mice with orthotopically developed HCT116 tumor in cecum, in monotherapy or in combination with metformin (MetF), already shown to enhance the hsTRAIL anti-tumor activity in subcutaneous CRC model. Oral administration of L.lactis (hsTRAIL+) resulted in significant progression of HCT116 tumors and shortening of the colon crypts. Secretion of hsTRAIL in the colon was accompanied by infiltration of the primary tumor with M2-macrophages, while MetF promoted transient colonization of the gut by L.lactis . Our study indicates that L.lactis bacteria after oral administration enable delivery of biologically active hsTRAIL to colon, however its potential therapeutic effect in CRC treatment is abolished by its pro-tumorigenic signalling, leading to the recruitment of M2-macrophages and tumor growth promotion.
... The implantation success rate was high (87% vs. 67% by Greenlee et al. (54) and 65% by Evans et al. (58)) and the distant metastasis was biologically relevant in this model (liver (15%) and intra-abdominal lymph node (20%) metastases in human MSI-H CRC) (45). Orthotopic CRC models have been serving as valuable tools for studying genes involved in metastasis (56) and evaluating the efficacy of immune checkpoint blockade therapies (59) and other therapies (e.g., recombinant methioninase to target the methionine (MET)-dependent cancers (62)). The model described here is the first orthotopic model of MMRd/MSI-H intestine cancer, and well suited for studying MMRd/MSI-H cancer biology and preclinically testing vaccines and immunotherapies in combination with targeted therapies for MSI-H tumors. ...
Background
Genome integrity is essential for the survival of an organism. DNA mismatch repair (MMR) genes (e.g., MLH1, MSH2, MSH6 , and PMS2 ) play a critical role in the DNA damage response pathway for genome integrity maintenance. Germline mutations of MMR genes can lead to Lynch syndrome or constitutional mismatch repair deficiency syndrome, resulting in an increased lifetime risk of developing cancer characterized by high microsatellite instability (MSI-H) and high mutation burden. Although immunotherapy has been approved for MMR-deficient (MMRd) cancer patients, the overall response rate needs to be improved and other management options are needed.
Methods
To better understand the biology of MMRd cancers, elucidate the resistance mechanisms to immune modulation, and develop vaccines and therapeutic testing platforms for this high-risk population, we generated organoids and an orthotopic mouse model from intestine tumors developed in a Msh2-deficient mouse model, and followed with a detailed characterization.
Results
The organoids were shown to be of epithelial origin with stem cell features, to have a high frameshift mutation frequency with MSI-H and chromosome instability, and intra- and inter-tumor heterogeneity. An orthotopic model using intra-cecal implantation of tumor fragments derived from organoids showed progressive tumor growth, resulting in the development of adenocarcinomas mixed with mucinous features and distant metastasis in liver and lymph node.
Conclusions
The established organoids with characteristics of MSI-H cancers can be used to study MMRd cancer biology. The orthotopic model, with its distant metastasis and expressing frameshift peptides, is suitable for evaluating the efficacy of neoantigen-based vaccines or anticancer drugs in combination with other therapies.
... Subcutaneously mimicking a tumor that from origin grows in another organ is less ideal as the invasive potential of the tumor is limited due to the presence of s.c. connective tissue, therefore creating a different tumor microenvironment (most likely also influencing the function/role of tissue resident immune cells) (52). This can impact the translatability of these models for immunotherapeutic research. ...
... This can impact the translatability of these models for immunotherapeutic research. For example, in preclinical melanoma models the tumor location was shown to influence the recruitment of tissue-specific tumor-associated macrophages (51,52). Orthotopically inoculated tumors in immune-competent mice can surmount some of these limitations. ...
Immune checkpoint inhibitors (ICI) have been revolutionary in the field of cancer therapy. However, their success is limited to specific indications and cancer types. Recently, the combination treatment of ICI and chemotherapy has gained more attention to overcome this limitation. Unfortunately, many clinical trials testing these combinations have provided limited success. This can partly be attributed to an inadequate choice of preclinical models and the lack of scientific rationale to select the most effective immune-oncological combination. In this review, we have analyzed the existing preclinical evidence on this topic, which is only limitedly available. Furthermore, this preclinical data indicates that besides the selection of a specific drug and dose, also the sequence or order of the combination treatment influences the study outcome. Therefore, we conclude that the success of clinical combination trials could be enhanced by improving the preclinical set up, in order to identify the optimal treatment combination and schedule to enhance the anti-tumor immunity.
... 203 A serious drawback using subcutaneous or orthotopic PDX in modeling CRC is that the microenvironment in an immuno-deficient mouse is significantly different from that of a tumor growing in an otherwise normal gastrointestinal tract. 197,204 Immune-deficient mice are prone to metastasis, and this has been taken advantage of for the testing of combinations of many drugs, but a major aim of personalized medicine has been to develop chemotherapeutic regimens such as checkpoint inhibitors that are less deleterious to the patient. To test the effectiveness of an immune checkpoint inhibitor in a mouse, there must be an immune system to release. ...
Mouse models of colorectal cancer (CRC) have been crucial in the identification of the role of genes responsible for the full range of pathology of the human disease and have proved to be dependable for testing anti-cancer drugs. Recent research points toward the relevance of tumor, angiogenic, and immune microenvironments in CRC progression to late-stage disease, as well as the treatment of it. This study examines important mouse models in CRC, discussing inherent strengths and weaknesses disclosed during their construction. It endeavors to provide both a synopsis of previous work covering how investigators have defined various models and to evaluate critically how researchers are most likely to use them in the future. Accumulated evidence regarding the metastatic process and the hope of using checkpoint inhibitors and immunological inhibitor therapies points to the need for a genetically engineered mouse model that is both immunocompetent and autochthonous.
... To further test efficacy in a model with increased heterogeneity and a more realistic tumor microenvironment [37,38], we employed an orthotopic pancreatic cancer model using cancer cells (CHX2000) derived from genetically modified KPC mice with pancreatic cancer to investigate if V-aCD3 Mu was efficacious in mice with established solid tumors. On day 22, we observed statistically significant inhibition of tumor growth in the V-aCD3 Mu treatment group (p = 0.0079), with a particularly strong effect in three out of five mice (Fig. 4B, S4A, and S4B). ...
Background
The malaria protein VAR2CSA binds oncofetal chondroitin sulfate (ofCS), a unique chondroitin sulfate, expressed on almost all mammalian cancer cells. Previously, we produced a bispecific construct targeting ofCS and human T cells based on VAR2CSA and anti-CD3 (V-aCD3 Hu ). V-aCD3 Hu showed efficacy against xenografted tumors in immunocompromised mice injected with human immune cells at the tumor site. However, the complex effects potentially exerted by the immune system as a result of the treatment cannot occur in mice without an immune system. Here we investigate the efficacy of V-aCD3 Mu as a monotherapy and combined with immune checkpoint inhibitors in mice with a fully functional immune system.
Methods
We produced a bispecific construct consisting of a recombinant version of VAR2CSA coupled to an anti-murine CD3 single-chain variable fragment. Flow cytometry and ELISA were used to check cell binding capabilities and the therapeutic effect was evaluated in vitro in a killing assay. The in vivo efficacy of V-aCD3 Mu was then investigated in mice with a functional immune system and established or primary syngeneic tumors in the immunologically “cold” 4T1 mammary carcinoma, B16-F10 malignant melanoma, the pancreatic KPC mouse model, and in the immunologically “hot” CT26 colon carcinoma model.
Results
V-aCD3 Mu had efficacy as a monotherapy, and the combined treatment of V-aCD3 Mu and an immune checkpoint inhibitor showed enhanced effects resulting in the complete elimination of solid tumors in the 4T1, B16-F10, and CT26 models. This anti-tumor effect was abscopal and accompanied by a systemic increase in memory and activated cytotoxic and helper T cells. The combined treatment also led to a higher percentage of memory T cells in the tumor without an increase in regulatory T cells. In addition, we observed partial protection against re-challenge in a melanoma model and full protection in a breast cancer model.
Conclusions
Our findings suggest that V-aCD3 Mu combined with an immune checkpoint inhibitor renders immunologically “cold” tumors “hot” and results in tumor elimination. Taken together, these data provide proof of concept for the further clinical development of V-aCD3 as a broad cancer therapy in combination with an immune checkpoint inhibitor.
