Mouse-Derived Isograft (MDI) In Vivo Tumor Models I. Spontaneous sMDI Models: Characterization and Cancer Therapeutic Approaches

Syngeneic in vivo tumor models are valuable for the development and investigation of immune-modulating anti-cancer drugs. In the present study, we established a novel syngeneic in vivo model type named mouse-derived isografts (MDIs). Spontaneous MDIs (sMDIs) were obtained during a long-term observation period (more than one to two years) of naïve and untreated animals of various mouse strains (C3H/HeJ, CBA/J, DBA/2N, BALB/c, and C57BL/6N). Primary tumors or suspicious tissues were assessed macroscopically and re-transplanted in a PDX-like manner as small tumor pieces into sex-matched syngeneic animals. Nine outgrowing primary tumors were histologically characterized either as adenocarcinomas, histiocytic carcinomas, or lymphomas. Growth of the tumor pieces after re-transplantation displayed model heterogeneity. The adenocarcinoma sMDI model JA-0009 was further characterized by flow cytometry, RNA-sequencing, and efficacy studies. M2 macrophages were found to be the main tumor infiltrating leukocyte population, whereas only a few T cells were observed. JA-0009 showed limited sensitivity when treated with antibodies against inhibitory checkpoint molecules (anti-mPD-1 and anti-mCTLA-4), but high sensitivity to gemcitabine treatment. The generated sMDI are spontaneously occurring tumors of low passage number, propagated as tissue pieces in mice without any tissue culturing, and thus conserving the original tumor characteristics and intratumoral immune cell populations.


Doc. S1-sMDI: sMDI -Histological and Pathological Analysis p 11
The document summarizes detailed histopathological analysis of primary and derived tumors of various sMDI models inclusive respective large microphotographs

Whole transcriptome shotgun sequencing-based (RNA-Seq) Expression pattern of tyrosine kinase receptor (TKR) gene family
RNA-isolation and RNA-Seq were performed by StarSeq, Mainz, Germany as whole transcriptome shotgun sequencing analysis from samples of sMDI JA-0009 and cMDI JA-2011 and JA-2042. RNA-Seq comparisons were performed based on respective FPKM (fragments per kilobase million) values. Since we could not yet determine the definite tissue of origin of the outgrowing MDI tumors, it was not possible to compare tumor gene expression with its respective -unfortunately unknown-normal tissue equivalent. Thus, we performed these experiments as a means of proof of principle, using the example of three gene families which are related to tumor malignancy or anti-tumoral immune response in three different MDIs.
Table S1a summarizes duplicate FPKM values of eighteen genes of tyrosine kinase receptor (TKR) gene family separately determined from single RNA-Seq experiment raw data from two distinct, frozen stored tumor samples of one single passage of JA-0009, JA-2011 or JA-2042 MDI each.
Duplicate FPKM values do reflect an objective gene expression pattern within the individual MDI models. Up to 1,000fold different expression patterns comparing FPKM values of individual TKR genes were observed. For example, anaplastic lymphoma kinase, Alk (mean FPKM: 0.126), showed very weak (less than 1,000-fold lower) expression, compared with AXL receptor tyrosine kinase, Axl (mean FPKM: 167.732), whereas erb-b2 receptor tyrosine kinase 4, Erbb4, was not expressed at all in sMDI JA-0009 model. However, since normal tissue equivalents of MDIs are unknown, one only could speculate about putative malignancy-dependent variances.

Doc. S1 -sMDI: sMDI -Histological and Pathological Analysis
The document summarizes detailed histopathological analysis of primary and derived tumors of various sMDI models inclusive respective large microphotographs Doc. S1-sMDI Pathological investigations on index and recipient mice with the present sMDI tumors:   Index tumor JA-0011 (Fig. 2) was classified as a histiocytic sarcoma (HS) or histiocyteassociated lymphoma (HAL), as localized in several tissues (liver, gut, kidney, spleen -see Figure 1 to Figure 5), with both histiocytic and lymphocytic components. Re-transplanted subcutaneous tumors, 0339-17, 4007-16, or 0057-17 also invaded spleens e.g. 1298-16 ( Figure 6) and/or livers e.g. 0052-17 (Figure 7) of recipient mice, with either lymphoblastic (e.g. 1298-16) or histiocytic predominant differentiation . These features are topographical and histological characteristics of these hemolymphopoietic neoplasms. The malignant proliferation is characterized by sheets and bundles of pale macrophage-like histiocytic cells, which progressively compress, invade and replace the hepatic parenchyma and by the presence of many multinucleate giant cells (long black arrows). There is also relatively significant associated inflammation and/or recruited mixed leukocytes, a majority of which is characterized by histiocytes/macrophages infiltrates cytologically within normal limits, a few of which in a necrotic/degenerated form (small black arrow in the upper left quadrant area). Accompanying this histiocytic and macrophagic proliferation, are significant infiltrates of polymorphonuclear (PMN) leukocytes, associating both neutrophils (blue arrows) and some eosinophils (red arrows). On this particular high magnification view centered on a central vein (CV), the neoplastic proliferation (cell type) is not particularly obvious. The neoplastic process is chronic (probably relatively indolent/slow growing neoplasm), as judged by the associated florid fibrosis accompanying the predominantly granulomatous-like inflammation (see arrowhead ).
Morphological diagnosis: Histiocytic Sarcoma/ Histiocyte-associated lymphoma.  A proliferative infiltrate similar to liver (see Figure 2 and Figure 3) and kidney is noted (blue stars), markedly thickening the serosal surface of a cavitary organ most compatible with oviduct adjacent to ovary. This proliferative tissue induces marked dilatation of blood and lymph vessels (blue arrows). SM = smooth muscle layer of the oviduct.
Consequently, it may be surmised that the intestinal mass was most probably a heavily invaded ovary (see Figure 1  The spleen is grossly enlarged (see thumbnail below) and the red pulp is diffusely invaded with sheets of histiocytic and cells with occasional to numerous giant multinucleate cells. The white pulp is also invaded and partially erased by the neoplastic process. Contrast with case in Figure 6, where a malignant lymphoma is present in the spleen of case 1298-16.

JA-0013 sMDI: Malignant Lymphoma (ML)
The phenotype of tumor JA-0013/ as exemplified by specimens 0013-14 (index case) and mice 1562-16, 0026-17, 0027-17 and 0028-17 (Fig. 2) was most consistent with malignant lymphoma, which was probably of B-cell lineage, as judged by the presence of infiltration of blastic cells with immunoblastic or plasmacytic differentiation (Figure 10) plasma and Mott cells (plasma cells containing Russell bodies) and the presence of large histiocytoid cells (see Figure 10). It had a remarkable phenotypic stability (see Figure 11 and Figure 12). One daughter re-transplanted tumor studied microscopically, 0027-17, had an intriguing finding, non-neoplastic giant multinucleate giant cells, leading to suspect a possible complication with an opportunistic organism (see Figure 13).
Gross aspects were also typical of malignant lymphoma (see Figure 9) There is markedly enlarged liver, 2 masses in pulmonary hilus in the thorax (behind the diaphragm) and a markedly enlarged spleen This specimen from pulmonary hilus/diaphragmatic masses exhibits sheets of malignant midsized blastic lymphocytes with presence of large, 50-70 µm diameter atypical cells with a pale glassy eosinophilic cytoplasm (black arrows). The sheets are irregularly infiltrated with smaller cells with a stem cell morphology, which could be of neoplastic origin or myeloid in origin (related to a response to anemia) and with plasmacytic cells, either neoplastic like (blue arrows) suggesting plasmacytic differentiation, or typical plasma cells with Russel bodies (Mott cells -see red arrows). Tumor infiltrating lymphocytes and other normal host inflammatory cells cannot be appraised without special molecular pathology techniques based on HEstained sections and morphology alone. Tissue of origin: very likely lymph node (presence of lymphatic vessels -not present in the field above) and lymphatic sinuses filled with small lymphocytes (see red arrows -architecture partially preserved) There is partial erasing of lymphoid architecture by homogenous sheets of round cells most compatible with proliferating (blastic) lymphocytes and scattered rare multinucleate giant cells (MGCs) that have large empty vacuoles or vacuoles containing pale amorphous pale gray material (arrows) and are not considered to be neoplastic.
This finding of non-neoplastic giant multinucleate giant cells is intriguing, and leads to suspect a possible complication with an opportunistic organism.
B cell markers would be useful to characterize the proliferation. In previous page: The proliferation is composed of homogenous sheets of small (~ 5 µm in dimeter) to mid-sized (~ 7 µm) well differentiated lymphocytes, which are diffusely invading into the subcutis or a pre-existing lymph node. Inset: cells are 7-8 µm and there are also evidence of macrophages with phagocytized apoptotic debris ("starry sky" aspect)..

Morphological diagnosis: Malignant lymphoma, small cells, intestine
Source files: This specimen is in the subcutis adipose tissue, with diffuse invasion. The lymphoma cells are small/medium sized, measuring 7-8 µm and there is also as in index case macrophages with phagocytized apoptotic debris ("starry sky" aspect) -see red arrows.
The morphological similarity with the index case (see Figure 18) is striking.

Morphological diagnosis: Malignant lymphoma, small cells, intestine
Source file:

JA-0021 sMDI: Malignant Lymphoma (ML)
In the case of tumor JA-0021 (Fig. 2), starting from various suspicious tissues, an enlarged lymph node from female C3H/HeJ mouse was re-transplanted. Although weakly growing in primary recipient SCID/bg mouse 1205-16. The para-cortical region of this sub-cutaneous lymph node has an early invasion with homogenous sheets of small lymphocytes (see black arrows to bracket the foci). Although an atypical lymphocytic immune response cannot be strictly rule out on an isolated case, the context suggests these early sheets of cells to represent malignant lymphoma. The surrounding tissue is mammary gland tissue in adipose tissue of subcutis (panniculus). Inset: The lymphocytes are small and homogenous in morphology, measuring 5-7 µm in diameter.
Morphological diagnosis: Malignant lymphoma, panniculus/sub-cutis, early invasion  There is partial to complete erasing of the normal lymphoid architecture of a lymphoid organ by homogenous sheets of small (5 to 7 µm) round cells (see inset for details), most compatible with blastic lymphocytes, which invade the adjacent tissues, here most probably the adipose tissue of the subcutis and/or the skin (black arrows and blue arrow below in B showing mammary gland tissue). There is evidence of mitoses and apoptosis (inset of A: red arrows). Further characterization of the lymphoma would be an advantage.
Morphological diagnosis: Malignant lymphoma, panniculus/sub-cutis, ♀ SCID bg/bg. The index animal had a subcutaneous mass in the lymph node region and a markedly enlarged spleen, suggesting a malignant lymphoma (see Figure 23). Histology showed the "lymph node" tumor to be a transmissible neoplasia of epithelial glandular origin ( Figure 27 and Figure 24) located in the skin/subcutis with possible anatomical localization in/adjacent to a lymph node or other organ (origin could be apocrine sweat gland, mammary gland etc.). It was an aggressive neoplasm with abundant epidermoid (Figure 25 -actually predominant feature of the daughter transmitted neoplasm 1286/16 see Figure 28 and Figure 29) and spindle cell differentiation ( Figure 26). As the spleen was not sampled for microscopic evaluation, the presence of metastases of the epithelial neoplasm (carcinoma) could not be verified, and therefore a concomitant neoplasm of the hemolymphopoietic lineage (leukemia or lymphoma) cannot be strictly ruled out in this particular case.    There is presence of cysts with papillary proliferations and many tubular profiles, with abundant evidence of secretion materials and necrotic debris, demonstrating the glandular origin of this aggressive neoplasm (black arrows). The tumor is clearly cutaneous/sub-cutaneous (blue arrow). The proliferation is composed of both myoepithelial (arrowheads ) and glandular elements (red arrows) with preserved ductular elements (black arrow). These elements are well differentiated and benign looking. This neoplasm was however transmitted.  The neoplasm 005-17 is similar to neoplasm 0023-14 from the subcutis (see Figure 36), with a somewhat more papillo-tubular than tubular and less desmoplastic morphology Source file: High magnification is more cellular than the index tumor from the subcutis (see Figure 36). Notice the tumor aspect is very different from the renal tumor from the index case 0023-14 (see Figure 35) but similar morphologically to all other tumors from the lineage (see Figure  38, Figure 40, Figure 41 and Figure 42), including the subcutaneous index mammary cancer (Figure 36). High magnification is identical to the lineage tumor 0005-17 ( Figure 38). There is no evidence of normal renal tissue on the section examined. The tubulopapillary pattern is present, as well as the host immune response (see lower inset of the green square)  The index case was previously studied in phase study TPL Path Labs TPL780-17.
The malignant proliferation is characterized by epithelial cell proliferation forming tubules and papillar (glandular) differentiation (black arrows), developed in a 6.8-millimeter long, multilocular cystic cavity (arrowhead ). Accompanying this epithelial malignant tumor of glandular origin are slight to moderate, stromal mononuclear leukocyte infiltrates, associating both small lymphocytes (blue arrows) and some macrophages. A lymphatic vessel is markedly dilated (red arrow).

Low magnification of tumor aspect overview (thumbnail):
Low magnification view of tumor case ID # 0547. The neoplasia is developed in 7x4-mm cyst, with a multi-locular pattern (development of secondary cysts -see arrowheads ).

Cyst
The red rectangle indicates the part of the cyst wall, which is depicted above at higher magnification.  Figure 41). Stromal infiltration with mononuclear cells is of similar nature and degree (blue arrows). Stroma also has similar levels of tumor-infiltrating lymphocytes (TIL). Although rather well differentiated, this neoplasm is clearly malignant, with good seeding potential, with also compression related dilated lymphatic vessels (red arrow). Cytological features of this tumor and the ones from all JA0023 derived tumors are very similar, near identical. The neoplasm is strikingly invasive in the adjacent stroma (not shown).

Skin, nipple
The red rectangle indicates the part of the tumor, which is depicted above at higher magnification. At sub-gross examination, this ~ 3.5-mm long neoplasm is developed in several multilobulated dilated or cystic ( ) spaces, composed of tubule and papillary proliferations. This tumor from 0055-17 is most probably derived from the index lung Adk. The clear cell aspect most probably is to be related to surfactant secreting type II pneumocytes -IHC would be useful to prove this hypothesis. Standard HE histology cannot prove it.
This situation is a frequent encounter, when the sites of grafting is different from the original organ of origin. An orthotopic (matching site in the lung) engraftment would be desirable if one is willing to recreate the original morphology (see the recent publication in Reference 2 for the pancreatic cancer situation. In this particular case, if further used in research and development, orthotopic engraftments would be highly recommended, although heterotopic (ectopic) may be used, as long as the presence of key characteristics such as the surfactant and areas of alveolar morphology can be demonstrated

Fig. S1-sMDI -Efficacy of anti-ICPI antibodies in seven established syngeneic standard mouse tumor models
The tumors cells of colon carcinoma MC38-CEA, colon carcinoma CT26.WT, lung carcinoma LL/2, melanoma Clone M3, mamma carcinoma 4T1, renal carcinoma RENCA, and melanoma B16.F10 were each implanted subcutaneously into mice. Animals were randomized at tumor volumes of 100 -200 mm 3 between days 5 -13 according to the respective model into 8 mice per group. Mice were treated three times (dotted lines) i.p. with anti-ICPI antibodies and PBS as vehicle control: vehicle -blue (10 ml/kg), anti-mPD-1 -green (10 mg/kg), and anti-mCTLA-4 -red (10 mg/kg). Tumor volumes are shown as growth curve (curve chart), mean of groups (bar graphs), and individual values of single mouse per group (dot plots). Probability (P) was tested with parametric unpaired t test (GraphPad Prism 5.04) compared to PBS vehicle control. Differences were determined as not significant with ns > 0.050 and significant * with p < 0.050, ** with p < 0.010, or *** with p < 0.001.