Mouse-Derived Isograft (MDI) In Vivo Tumor Models II. Carcinogen-Induced cMDI Models: Characterization and Cancer Therapeutic Approaches

In this second study, we established syngeneic in vivo models named carcinogen-induced mouse-derived isografts (cMDIs). Carcinogen-induced tumors were obtained during short-term observation (3–9 months) of CBA/J mice treated with various administration routes with 3-methylcholanthrene (MCA) or N-methyl-N-nitrosourea (MNU) as carcinogens. During necropsy, primary tumors and suspicious tissues were assessed macroscopically and re-transplanted (in PDX-like manner) into sex-matched syngeneic animals. Outgrowing tumors were histologically characterized as either spinocellular carcinoma (1/8) or various differentiated sarcomas (7/8). Growth curves of four sarcomas showed striking heterogeneity. These cMDIs were further characterized by flow cytometry, RNA sequencing, or efficacy studies. A variable invasion of immune cells into the tumors, as well as varying expression of tyrosine kinase receptor, IFN-γ signature, or immune cell population marker genes could be observed. Immune checkpoint inhibitor treatment (anti-mPD-1, anti-mCTLA-4, or a combination thereof) showed different responses in the various cMDI models. In general, cMDI models are carcinogen-induced tumors of low passage number that were propagated as tissue pieces in mice without any tissue culturing. Therefore, the tumors contained conserved tumor characteristics and intratumoral immune cell populations. In contrast to the previously described spontaneous MDI, carcinogen induction resulted in a greater number of individual but histologically related tumors, which were preferentially sarcomas.


Doc. S1-cMDI: cMDI -Histological and Pathological Analysis
The document summarizes detailed histopathological analysis of primary and derived tumors of various cMDI models inclusive respective large microphotographs    The sarcomatous tissue fully invades the dermis and widely dissects out the preexisting dermis (left inset), eliciting focal epidermal hyperplasia and ulceration at tumor center (black arrows). This is one of the 3 fragments examined  Sarcoma composed of interlacing bundles alternating longitudinal (black arrows) and transverse bundles (black arrows) or long, rather well differentiated spindle-shaped cells.
There is no ischemic necrosis on the section examined.
The red rectangle indicates the part of the tumor, which is depicted above.
Notice the homogenous sarcomatous tissue, infiltrating the preexisting skeletal muscles (red arrow) on the edge of the sample.
This detail of the neoplastic proliferation is located in the green rectangle of the topographical photo above The neoplastic cells exhibit low pleomorphism but have numerous infiltrating lymphocytes (blue arrow) and deeply invade between atrophic muscle fibers (red arrows). The invaded tissue type is unrecognizable (it invades muscle and fat). The sarcoma is composed of poorly intertwined bundles of blastic deeply basophilic roughly spindle-shaped cells (black arrows). When compared to the originator neoplasm (see Figure 10), the main differentiating feature is presence of wide ischemic necrosis indicative of inadequate vascularization (red arrows). Lymphocyte infiltration is very dense.
The red rectangle indicates the part of the tumor, which is depicted above in Figure 11.
Notice the homogenous sarcomatous deeply basophilic tissue, surrounding the central frankly paler and more eosinophilic area of ischemic necrosis (red arrow), and deeply invading/ infiltrating the pre-existing fat tissue (white arrows).
This detail of the neoplastic proliferation is located in the green rectangle of the topographical photo above Neoplastic cells exhibit relatively low pleomorphism, occasionally prominent nucleoli (red arrow) rare mitoses and quite numerous infiltrating small lymphocytes (blue arrows). Mitoses are numerous (not shown). A A: main mass of the index tumor showing 2 distinct cell density aspects, a higher more basophilic aspect (black arrows) and a lower paler and pinker aspect (blue arrows) corresponding to lower cell density with also possibly paler cytoplasm. In addition there are 2 small areas with dilated vessels containing red blood cells, mimicking an hemangioma or hemangiosarcoma differentiation (see red arrows and red rectangle in previous page) Also, notice artefactual cracks in central areas of the sample (white arrows), suggesting lack of significant extracellular matrix.
B: Sub-gross view of grafted JA-2042 into 124-17 mouse. The tissue similarity at this magnification is striking. Importantly, there are vascular profiles (see red arrow There is also hemangiosarcoma differentiation, indicating remarkable phenotype stability (see red arrows and red rectangle). IHC is strongly advised here to document possible vascular differentiation.
Notice artefactual cracks in central areas of the sample (white arrows), suggesting lack of significant extracellular matrix. The adipose tissue of the panniculus is diffusely invaded by bundles of spindle-shaped cells, rather well differentiated, and of moderate anaplasia (see below; black arrows). Based on these samples, it is impossible to state whether the sarcoma NOS simply invades the panniculus or in fact exhibit liposarcomatous differentiation. Based on the normal aspect of the adipocytes, it is most likely that it is a sarcoma invading pre-existing normal adipose tissue?
Morphological diagnosis: Sarcoma NOS, MNU-induced, CBA/J mouse. The invaded tissue type is unrecognizable. The sarcoma is composed of interlacing bundles of blastic poorly differentiated spindle-shaped cells (black arrows). When compared to the originator neoplasm (see Figure 14), there is an increased degree of anaplasia, more mitoses, with few bizarre giant cells (green arrows) and evidence of limited early ischemic necrosis, indicative of inadequate vascularization (red arrows). Morphological diagnosis: Sarcoma NOS, MNU-induced, CBA/J mouse, 0125-17.
The red rectangle indicates the part of the tumor, which is depicted above.
Notice the homogenous sarcomatous tissue, with visible, slightly paler and more eosinophilic ischemic necrosis (red arrow). The sarcomatous tissue is characterized by high variability of size and shape of cells and nuclei ("anisopoikilocytosis" and "anisopoikilokaryosis"), vesicular nuclei and multinucleated cells with condensed nuclei and/or abnormal /abortive mitoses or "mitonecrosis" (see black arrows) -the section has some artefactual cracks. This is an example of moderate to low differentiation, with a rather anaplastic aspect. This aspect is on the top specimen of Figure 18.

Tumor diagnosis (HE evaluation)with description
Other comments Sarcoma NOS, varying aspects, from well differentiated to moderately anaplastic; no giant cells -deeply invading the skin up to the upper dermis -many anaplastic features (abnormal mitoses, large vesicular nuclei) but no giant multinucleate cells The tumor tissue is characterized by interwoven basket weave bundles of rather well differentiated fibroblasts, which however finely invade and dissect the pre-existing sheets of normal adipocytes of the subcutis. This aspect is on the low specimen of Figure 18. Extracted from TPL Study Phase Number 811/17 A: This is an epithelial neoplastic proliferation from the skin, characterized by epidermal differentiation and keratin production, which induces intense inflammation, with a pronounced lymphoid component (arrowheads ), as well as fibrosis (green arrows).

A :
It is characterized by cords and nodular elements of keratinocytes (red arrows) with central laminar keratinization, often forming central cysts with occasional horn pearls (black arrow).
The neoplasm is entrapping a nerve (blue arrows).
Morphological diagnosis: Spinocellular carcinoma, skin, MCA-induced, CBA/J mouse.  The red rectangle indicates the part of the neoplasm, which is depicted above

B:
The vast majority of the tumor on the section examined is composed of desmoplastic and highly inflamed stroma (green arrow).  The inflammatory cells composing the interface of intact and degenerated neoplastic tissue 16 (with hemorrhage) is mainly composed of intact and degenerated granulocytes, with focal 17 presence of mononuclear cells (see inset below next page). In central ischemic necrosis there 18 is associated red blood cell extravasation (red arrows). Areas of necrosis are numerous and coalescing with hemorrhage and abundant leukocytes: 25 Extracted from TPL Study Phase Number 811/17 26 Detail of the Figure 25 depicting the cytological details of the inflammatory infiltration at the interface of the intact and degenerated neoplastic tissue, with mainly intact and degenerated granulocytes (red arrow). Mononuclear cells suggesting monocytes and/or lymphocytes are relatively rare (blue arrow)