Transplantable Tumor Cell Line Research Articles

Intratumoral BO-112 in combination with radiotherapy synergizes to achieve CD8 T-cell-mediated local tumor control

BackgroundRadioimmunotherapy combines irradiation of tumor lesions with immunotherapy to achieve local and abscopal control of cancer. Most immunotherapy agents are given systemically, but strategies for delivering immunotherapy locally are under...

Mouse Preclinical Cancer Immunotherapy Modeling Involving Anti-PD-1 Therapies Reveals the Need to Use Mouse Reagents to Mirror Clinical Paradigms.

Simple SummaryImmune checkpoint inhibition has revolutionized clinical cancer care. As clinical use of these inhibitors increases, the ability to study the effects of these therapies in preclinical models becomes more important. This study highlights the need for using species appropriate reagents to properly evaluate the efficacy and toxicity of cancer immunotherapy in mouse models.Immune checkpoint inhibition (ICI) has emerged as one of the most powerful tools to reverse cancer induced immune suppression. Monoclonal antibodies (mAbs) targeting programmed cell death 1/programmed cell death ligand 1(PD-1/PD-L1) are FDA-approved and their clinical use is rapidly expanding. As opposed to the clinical paradigm, which can result in significant responses and toxicities, it has been difficult to reproduce these effects preclinically using mouse models. In large part, this is due to models, which employ rapidly growing ex vivo cultured transplantable tumor cell lines engrafted into young naïve inbred laboratory mice. However, another issue concerns the use and repeated application of xenogeneic reagents in mice (i.e., rat or hamster mAbs directed against mouse antigens at variance with clinical use of human or humanized mAbs). Building on our previous studies demonstrating that repeated administration of commonly used xenogeneic anti-PD-1 mAbs derived from both rat and hamster can induce fatal hypersensitivity in some tumor-bearing mice, we sought to compare these result with the effects of a mouse anti-mouse PD-1 mAb. Application of a murine anti-mouse PD-1 (clone: MuDX400) did not result in lethal anaphylaxis in the 4T1 tumor model. It also displayed superior antitumor effects in this and other tumor models, as it did not induce neutralizing antibody responses against the anti-PD-1 mAb, such as were observed when using xenogeneic anti-PD1 mAbs. These results demonstrate that more accurate preclinical modeling necessitates the use of mouse reagents mirroring the clinical scenario to ascertain long-term effects or toxicities, while avoiding xenogeneic responses, which do not occur clinically. Furthermore, these studies suggest a direct mechanism, whereby preclinical murine studies have often failed to recapitulate the clinical efficacy and toxicity of single agent checkpoint inhibition.

Abstract 3741: Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma

Abstract Transforming growth factor beta (TGF-β) isoforms are the primary mediators for TGF-β signaling via TGF-β receptors and downstream phosphorylation/dephosphorylation cascade. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Mouse 4T1 mammary carcinoma cell line is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone. Considering rather challenging preclinical evaluation of antitumor activity in tumor models, mouse 4T1 mammary carcinoma model has been widely used in the literature for evaluation of TGF-β antagonists. In this report, we describe the efficacy of ISTH0047 - a potent and selective TGF-β2 antisense oligonucleotide - in murine 4T1 primary tumors and lung metastasis following tumor cell injection into the mammary fat pad (orthotopic tumor model) of syngeneic Balb/c mice. Consistent with literature data generated with other classes of TGF-β antagonists (e.g., small-molecule kinase inhibitors, antibodies or TGF-β trap agents), although limited antitumor activity was demonstrated on primary tumor growth, marked and statistically significant decrease of lung metastasis number was observed upon subcutaneous administrations of ISTH0047. In addition, side by side comparison with murine surrogates of CTLA-4 or PD-1 antibodies indicated similar efficacy of all test items on lung metastasis in this model. Taken together, these encouraging results pave the way for in-depth preclinical evaluation of both ‘seed and soil’ theory and efficacy of combination regimen (immunomodulation) for better clinical outcome. Citation Format: Katja Wosikowski, Kathy Hasenbach, Jutta Petschenka, Diana Barea Roldan, Sebastian Kreiter, Ugur Sahin, Guillaume Serin, Julie-Orlane Redon, Marc Hillairet de Boisferon, Francis Bichat, Hanna Kohonen, Michel Janicot. Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3741.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 5323: Distinct roles of IL-17A in inflammation-induced tumor development and tumor immunosurveillance

Abstract While chronic inflammation is considered to be a critical component of tumorigenesis, the immune system can also prevent tumor development and/or growth. IL-17A and IL-17A secreting T cells, termed Th17 mediate inflammation via activation of a wide range of downstream inflammatory mediators that contribute to chronic inflammatory diseases. It also has been suggested that Th17 can be a constituent in tumor immunosurveillance since tumor-specific Th17 has been shown to eradicate established tumor. In the present study, we addressed the effects of inflammation/immunity driven by IL-17A on a primary tumor development model using a chemical carcinogen 3′-methylcholanthrene (MCA). We found that IL-17A deficient (IL-17A−/−) mice were protected from the primary tumor development by MCA. Paradoxically, the growth of primary tumors developed in IL-17A−/− mice was faster than that of tumors in WT (C57BL/6) mice. Although tumor cell lines derived from WT and IL-17A−/− mice grew equally in immunocompromised (T, B cell deficient C. B-17 scid) mice, those from IL-17−/− mice were easily rejected in C57BL/6 mice as compared to those from WT mice. Consistent with this, tumor growth of B16 melanoma, a non-immunogenic transplantable tumor cell line, was enhanced in IL-17A−/− mice. The apparently contrasting effects of IL-17A on tumor growth and development may reflect difference in cells producing IL-17A, namely cells mediate innate or acquired immunity. We transferred CD4+ T cells from WT mice into IL-17A−/−. While the transfer with CD4+ T cells from WT mice did not affect the primary tumor development in IL-17A−/− mice, the growth of primary tumors in IL-17A−/− mice transferred with WT CD4+ T cells was significantly suppressed. These results indicate that IL-17A promotes tumorigenesis possibly by enhancing inflammation and IL-17A produced by CD4+ T cells contributes to the immune surveillance to the emerging tumors as well. Overall, these results not only demonstrate that a single cytokine, IL-17A, plays contrasting role in tumor development and growth, but also indicate that immune inflammation-driven tumorigenesis and tumor immunosurveillance coexist in the same host. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5323.

Low Surface Expression of B7-1 (CD80) Is an Immunoescape Mechanism of Colon Carcinoma

Artificially enforced expression of CD80 (B7-1) and CD86 (B7-2) on tumor cells renders them more immunogenic by triggering the CD28 receptor on T cells. The enigma is that such B7s interact with much higher affinity with CTLA-4 (CD152), an inhibitory receptor expressed by activated T cells. We show that unmutated CD80 is spontaneously expressed at low levels by mouse colon carcinoma cell lines and other transplantable tumor cell lines of various tissue origins. Silencing of CD80 by interfering RNA led to loss of tumorigenicity of CT26 colon carcinoma in immunocompetent mice, but not in immunodeficient Rag-/- mice. CT26 tumor cells bind CTLA-4Ig, but much more faintly with a similar CD28Ig chimeric protein, thus providing an explanation for the dominant inhibitory effects on tumor immunity displayed by CD80 at that expression level. Interestingly, CD80-negative tumor cell lines such as MC38 colon carcinoma and B16 melanoma express CD80 at dim levels during in vivo growth in syngeneic mice. Therefore, low CD80 surface expression seems to give an advantage to cancer cells against the immune system. Our findings are similar with the inhibitory role described for the dim CD80 expression on immature dendritic cells, providing an explanation for the low levels of CD80 expression described in various human malignancies.

Mouse 4T1 Breast Tumor Model

The 4T1 mammary carcinoma is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone The 4T1 tumor has several characteristics that make it a suitable experimental animal model for human mammary cancer. First, tumor cells are easily transplanted into the mammary gland so that the primary tumor grows in the anatomically correct site, as described in this unit. Second, as in human breast cancer, 4T1 metastatic disease develops spontaneously from the primary tumor. Also, the progressive spread of 4T1 metastases to the draining lymph nodes and other organs is very similar to that of human mammary cancer. In this unit, a protocol describes surgical removal of the primary tumor, so that metastatic disease can be studied in an animal setting comparable to the clinical situation where the primary tumor is surgically removed, and metastatic foci remain intact. Another advantage of 4T1 is its resistance to 6-thioguanine. This property enables precise quantitation of metastatic cells, even when they are disseminated and at sub-microscopic levels in distant organs, as described here.

Genetic Resistance to a Marek's Disease Transplantable Tumor Cell Line in Chicken Lines Selected for Different Immunological Characters

The tumor incidence and mortality of Marek's disease (MD) were determined for 471 progeny from four pairs of lines two-way selected for different immunological characters: graft versus host reaction (GVHR) competence, IgG levels, antibody response to rabbit serum albumin (RSA), and anaphylactic shock to BSA. All chicks were inoculated at 1 wk of age with 1 to 2 × 106 viable cells of a MD lymphoblastoid cell line, MDCC-MSB1-41C (41 C). Tumor incidence differed significantly between the high (H) and low (L) lines of each GVHR-, IgG-, RSA-, or BSA-selected group. There were also significant differences in mortality rates between H and L lines of all selected groups. The IgG-H, BSA-H, and RSA-H lines were more resistant than their respective L lines. The H line of the GVHR-selected lines was more susceptible than the L line. Tumor regression could be detected only in the IgG- and RSA-selected lines. No definite correlation could be found between genetic resistance to 41C and genetic resistance to MD virus.

Resistance of tumor cells to tumor necrosis factor

TNF-alpha is clearly an important mediator of in vitro tumor cell cytotoxicity induced by the activated macrophage. There are a number of other nonspecific mediators of tumor cell cytotoxicity. These include natural killer cells (51, 52), lymphokine-activated killer cells (53), and natural cytotoxic cells (54). The role that TNF-alpha may play in the cytotoxicity induced by these cell types has not been completely elucidated. Neither is it known what role, if any, TNF-alpha may play in major histocompatibility-restricted (T cell)-mediated tumor cell cytotoxicity. Just as in the case of the activated macrophage, activated cytotoxic T cells produce a number of mediators that inhibit the growth of tumor cells or that induce tumor cell cytotoxicity (55). The role that TNF-alpha plays in the whole process of the regulation of tumorigenesis will not become completely defined until an appropriate set of genetic experiments is completed which utilizes transplantable tumor cell lines selected specifically for resistance to this cytokine in in vivo tumor models. The prominance of TNF-alpha as a mediator of macrophage-induced tumor cell cytotoxicity makes it a candidate for analysis in studies of the early stages of tumorigenesis. We have chosen to study mechanisms of resistance to this monokine. Our results have shown that there are multiple pathways leading to resistance to TNF-alpha-induced tumor cell cytotoxicity. These pathways include the production of transforming growth factors by tumor cells and the amplified expression of certain oncogenes. Other pathways will undoubtedly become elucidated as we begin to define the molecular mechanisms giving rise to the resistant phenotype.

Synergistic and overlapping activities of tumor necrosis factor-alpha and IL-1.

TNF-alpha and IL-1 induce the production of PGE2 from human chondrosarcoma, fibrosarcoma, and carcinoma cell lines. When combined at sub-optimal concentrations, TNF-alpha and IL-1 synergistically stimulate PGE2 production. The synergy of TNF-alpha and IL-1 on the induction of PGE2 is partially neutralized by specific antibodies. In vitro, human rTNF-alpha is directly cytotoxic to several human transplantable tumor cell lines. These include a human carcinoma, human chondrosarcoma, and a human transformed fibroblast cell line. The cytotoxic effect of TNF-alpha was abrogated by a specific, neutralizing, polyclonal antibody. IL-1 had no direct cytotoxic effect on these cell lines; however, IL-1 enhanced the cytotoxic effects of TNF-alpha. The synergy of these two cytokines in the cytotoxic assay was neutralized by the addition of specific neutralizing antibodies.

Transplantable Primate Tumors Induced by Rous Sarcoma Virus. I. Induction of Tumors Transplantable Into Young Marmosets<xref ref-type="fn" rid="FN1">2</xref>

The tumors induced in white-lipped marmosets (Saguinus fuscicollis, S. nigricollis) by Rous sarcoma virus (RSV) of chicken origin (RSV-SR) were not transplantable to allogeneic hosts. In contrast, RSV rescued from these tumors (RSV-M) induced sarcomas that were transplantable to young but not to adult marmosets. The tumors induced by RSV-M and the transplants rapidly enlarged, metastasized to various organs, and killed the recipients 29-59 days post inoculation. Cell lines were readily established from all transplantable sarcomas. No virus expression was detected in transplantable tumor cell lines by electron microscopy or by biochemical and biological assays. However, RSV of the same subgroup as RSV-SR was rescued from both short-term and long-term tumor cell cultures by cocultivation with chicken embryo fibroblasts (CEF). The rescued viruses transformed marmoset cells 100-fold more efficiently than CEF cells, although CEF cells remained permissive for virus replication. Cytogenetic studies revealed extensive chromosome abnormalities in tumor transplants but not in RSV-M-induced sarcomas. All cell lines were hyperploid and contained structurally abnormal, large metacentric and telocentric chromosomes. Immunologic studies failed to detect group-specific (gs) antigen of the avian sarcoma-leukemia complex in either RSV-M-induced, transformed cells or tumor transplants. By complement-dependent cytotoxicity assays, with the use of marmoset anti-gs serum, RSV-associated antigen could be detected on the surfaces of tumor cells. No differences in the expression of this antigen existed between transplantable and nontransplantable marmoset sarcomas. All transplantable cell lines contained abnormal amounts of lipids and glycogen in comparison to RSV-SR-induced tumors and normal marmoset cell lines. The glycogen was associated with unique cytoplasmic membrane complexes and was surrounded by either single- or double-membraned vesicles.

Mode of action of calcium antagonists which alter anthracycline resistance

In transplantable tumor cell lines, a common mode of resistance to several natural products, including the anthracyclines, the Vinca alkaloids and actinomycin D, involves an outward transport process which limits drug accumulation to sub-lethal levels (1–5). This transport system can be inhibited, and drug responsiveness promoted, by calcium antagonists of two structural classes: papaverine analogs, e.g. verapamil, and dihydropyrimidines, e.g. nitrendipine (6–9). This report describes studies on the two classes of calcium antagonists named above. The data indicate that verapamil is a substrate for the outward transport system and promotes anthracycline accumulation via competition for exodus. Nitrendipine is not a substrate for this outward transport system and may act via a ‘chaotropic’ mechanism.

The effects of dichloromethylene diphosphonate on hypercalcemia and other parameters of the humoral hypercalcemia of malignancy in the rat Leydig cell tumor.

There is a high frequency of Leydig cell tumors associated with hypercalcemia in the aged Fischer 344 rat. We studied a transplantable tumor cell line (Rice D-6) which is associated with hypercalcemia, hypercalciuria, hypophosphatemia, renal phosphate wasting, increased urinary cyclic adenosine monophosphate (AMP) excretion, absence of bone metastases, increased osteoclastic bone resorption, and suppressed immunoreactive parathyroid hormone (iPTH) concentrations. We examined the ability of dichloromethylene diphosphonate (Cl2MDP) to lower serum calcium and decrease the parameters of increased bone resorption. We used this drug also as a pharmacologic tool to determine the relationship of hypercalcemia and increased bone resorption to the abnormalities in renal tubular function associated with the humoral hypercalcemia of malignancy.

The human tumor cloning assay in cancer drug development. A review.

Animal studies with transplantable tumor cell lines suggested that the sensitivities of the in vitro tumor cultures to certain anticancer drugs agreed with the drug sensitivities of the same tumors in vivo. Soft agar cloning techniques have been established for human myeloma and ovarian cancer cells. Refinement of the techniques now permits cloning of most human malignancies. Drug sensitivity studies have been conducted measuring the reduction in the number of tumor colonies formed. Clinical trials showed that approximately one half of patients whose cells demonstrate in vitro sensitivity to a drug will obtain a clinical response to the same drug. Patients whose cells appear resistant to a certain drug in vitro rarely respond to the drug clinically. In vitro drug sensitivity testing may eventually allow some individualization of chemotherapy. In addition it already appears that these techniques may be excellent for screening new drugs with specific anticancer potentials.