Abstract
Human tumor xenografts in immunodeficient mice have led to valuable insights into the biology of human cancer. The corresponding limitations and pitfalls of xenograft models have been ...described extensively. To tackle some limitations, we supplemented the murine tumor microenvironment (TME) with human stromal fibroblasts to mirror tumor-stroma cross-talk in vivo.
Non-small cell lung cancer (NSCLC) cell lines Calu-1 and H1437 were co-injected with different types of fibroblasts into NOG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice in 3 independent experiments (n = 4-6/group; 106 in total). Human Dermal Fibroblasts (HDFs), lung cancer associated fibroblasts (CAFs), corresponding normal fibroblasts (NFs) and NSCLC cells were implanted into the lungs of 4-6 week old recipient mice. The influence of extracellular matrix components was evaluated by using either a matrigel/collagen mixture or alginate capsules. Tumor load was determined via overall survival (OS) and flow cytometry (FC). When mice had to be sacrificed, organs were collected and analyzed by FC and patho-histological examination. In a subsequent experiment, mice were sacrificed and tumors analyzed at defined time points and similar analyses performed.
OS and metastatic pattern were similar in both cell lines when injected in mono-culture. OS of Calu-1 injected mice was not influenced when cells were co-injected with HDF but prolonged when co-injected with CAFs or NFs. In H1437 bearing mice, OS was significantly reduced when cells were co-injected with HDF or NF (Log-rank Mantel-cox test; p< 0.002 & 0.005), whereas CAFs had no influence on OS. Co-injection of HDF and Calu-1 enhanced the number of tumor cells in bronchoalveolar lavage fluid, whereas CAFs had the opposite effect (1way ANOVA for all following statistics, p< 0.0001). Consistently, the number of circulating Calu-1 cells was highest in co-culture with HDF and significantly lower in mice bearing Calu-1 and CAF or NF (p<0.0021). Co-injection of fibroblasts enhanced the H1437 tumor load in all compartments. Of note, the effect was not statistically significant. The use of alginate capsules was favorable compared to matrigel/collagen, improving fibroblast engraftment in all investigated organs. Furthermore, alginate capsules enhanced the number of circulating H1437 cells when co-injected with HDF as compared to matrigel/collagen (p<0.0001).
Our results demonstrate the major impact of fibroblasts on tumor cell behavior in a preclinical setting. With the successful co-cultivation of human fibroblast and NSCLC cells in vivo, it will be possible to study tumor-stroma interactions in a clinically relevant mouse model. Once validated by a compound screening approach, this model will help to reduce drug failure rates and contribute to a more efficient development of urgently needed novel anti-cancer treatments.
Citation Format: Eva Oswald, Vitor E. Santo, Albin Rudisch, Catarina Brito, Wolfgang Sommergruber, Helmut Dolznig, Julia B. Schüler. Co-injection of human fibroblasts significantly enhances tumorigenicity of orthotopically implanted human non-small cell lung cancer cells in immunocompromised mice. 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 630.
Enzyme replacement therapy (ERT) with recombinant human alglucosidase alfa (rhGAA) was approved in Europe in 2006. Nevertheless, data on the long-term outcome of infantile onset Pompe disease (IOPD) ...patients at school age is still limited.
We analyzed in detail cardiac, respiratory, motor, and cognitive function of 15 German-speaking patients aged 7 and older who started ERT at a median age of 5 months.
Starting dose was 20 mg/kg biweekly in 12 patients, 20 mg/kg weekly in 2, and 40 mg/kg weekly in one patient. CRIM-status was positive in 13 patients (86.7%) and negative or unknown in one patient each (6.7%). Three patients (20%) received immunomodulation. Median age at last assessment was 9.1 (7.0-19.5) years. At last follow-up 1 patient (6.7%) had mild cardiac hypertrophy, 6 (42.9%) had cardiac arrhythmias, and 7 (46.7%) required assisted ventilation. Seven patients (46.7%) achieved the ability to walk independently and 5 (33.3%) were still ambulatory at last follow-up. Six patients (40%) were able to sit without support, while the remaining 4 (26.7%) were tetraplegic. Eleven patients underwent cognitive testing (Culture Fair Intelligence Test), while 4 were unable to meet the requirements for cognitive testing. Intelligence quotients (IQs) ranged from normal (IQ 117, 102, 96, 94) in 4 patients (36.4%) to mild developmental delay (IQ 81) in one patient (9.1%) to intellectual disability (IQ 69, 63, 61, 3x <55) in 6 patients (54.5%). White matter abnormalities were present in 10 out of 12 cerebral MRIs from 7 patients.
Substantial motor, cardiac, respiratory, and cognitive deficits are frequent in IOPD long-term survivors who started ERT before 2016. The findings of this study can be valuable as comparative data when evaluating the impact of newer treatment strategies including higher enzyme dosage, immunomodulation, modified enzymes, or early start of treatment following newborn screening.
Abstract
The aim of this study was to determine the influence of the engraftment site on the tumor biology and drug sensitivity of a panel of hematological patient derived xenografts (PDX). PDX cells ...(3x10e6 cells/mouse) were injected intratibialy (i.t.), intrasplenal (i.s.) or subcutaneously (s.c.) into NOG (NOD/Shi-scid/IL-2Rγnull) mice. Tumor engraftment was determined by flow cytometry (FC) in bone marrow (BM), peripheral blood (PB) and spleen during the course of engraftment and at the end of a study. Overall survival (OS) served as an additional read-out. In 3 models sensitivity towards cytarabine (Cy) was evaluated. Our group has established 18 PDX of acute leukemia (16 AML, 1 ALL, 1 APL). 16/17 lines engrafted when injected i.t., 10/12 developed tumors after i.s. implantation and 13/14 established tumors post s.c. cell injection. Thus, the overall engraftment capacity was for most of the models not depending on the injection site. Nevertheless, some models could only be propagated in a specific setting: LEXFAM 2713, grew exclusively i.t. or i.s., whereas LEXFAM 2824 could be propagated solely when injected s.c.. The implantation site did influence tumor growth rate: Mean OS ranged from 151.4 (±25.21) days for i.t. to 89.2 (±16.82) days for s.c. propagation. I.s. transplanted mice had to be sacrificed after 91.9 (±17.33) days. The dissemination pattern of individual lines was affected by the injection site. In general, infiltration of the hematopoietic organs was higher when cells were engrafted i.t. or i.s.. Nevertheless, also s.c. implanted, AML cells infiltrated murine PB, spleen and BM, although not consistently and to a much lower extent. Interestingly, the expression pattern of the 6 investigated surface markers (CD45, CD3, CD34, CD33, CD38 & HLA-ABC) was not influences by the application route. Every model depicted its distinct expression pattern irrespective of the application route. Cy was highly active in 1 AML (LEXFAM 2531) and 1 ALL (LEXFAL 2665) model. OS was significantly prolonged in the s.c. as well as in the disseminated setting (p< 0.003, Log-rank (Mantel-Cox) test). Another AML line (LEXFAM 2734) depicted a less pronounced sensitivity towards Cy (p< 0.007, Log-rank (Mantel-Cox) test) both growing s.c. or i.t.. Thus, drug sensitivity was not influenced by the injection site of the leukemic cells. Of note, the PDX drug responses mimicked the responsiveness of the respective donor patient. Taken together the leukemia PDX panel represents the molecular diversity of the disease and mirrors sensitivity towards standard of care. Our recent careful investigation of the subcutaneous approach in these models highlights their suitability for this type of studies. This enhances the value of the platform as it combines the possibilities of a mid-throughput screening enabled by the subcutaneous approach with the general advantages of a patient-derived leukemia model.
Citation Format: Julia B. Schüler, Eva Oswald, Gabriele Greve, Dorothee Lenhard, Kerstin Klingner, Milena Pantic, Michael Luebbert. Influence of the injection site on the dissemination pattern and drug sensitivity of patient derived leukemia cells in vivo abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4821. doi:10.1158/1538-7445.AM2017-4821
Abstract
The field of cancer immunology is rapidly moving towards innovative therapeutic strategies. As a consequence the need for robust and predictive preclinical platforms arises just as well. The ...current project aims to establish a drug screening workflow bridging between innovative mouse models and clinical biomarker development. A total of 69 NOG (NOD/Shi-scid/IL-2Rγnull) mice were engrafted with CD34+ hematopoietic stem cells. Thereafter, tumor material from 11 different lung cancer patient derived xenograft models (NSCLC PDX) was implanted subcutaneously. Individual mice were treated with α-CTLA-4, α-PD-1 or the combination thereof. With n=1 per treatment arm and model the study design followed the screening approach of the single mouse trial (SMT). Infiltration of human immune cells was detected by flow cytometry (FC) and immunohistochemistry (IHC) in hematopoietic organs and tumor tissue. A computerized analysis for digitized whole-slide images of the samples was used to quantify the lymphocyte infiltration using color classification and morphological image processing techniques. All 3 treatment arms displayed a discrete activity pattern throughout the PDX panel. Tumor models with high tumor infiltrating lymphocyte (TIL) rates in the donor patient material tended to be more sensitive towards checkpoint inhibitor treatment as models with low rates. Numbers of TILs in the PDX detected by FC and IHC were significantly increased in the treatment groups as compared to control vehicle. In parallel, hematopoietic organs showed high (>25%) amounts of huCD45 cells in all groups and models. PDX models being sensitive towards checkpoint inhibitor treatment (responders) displayed a higher percentage of DAB+ nuclei in huCD45 IHC stains than non-responder models as determined by image analysis. Irrespective thereof, in responders as well as non-responders the treatment with checkpoint inhibitors enhanced the percentage of DAB+ nuclei. Whole-slide image analysis of the H&E stains revealed an increase of the stromal compartment proportion in the tumor tissue under treatment with checkpoint inhibitors in responder models. In non-responder models the ratio between tumor and stroma was not influenced by drug treatment. The use of PDX based humanized mouse models in a SMT format allows screening approaches in complex mouse models. The combination with a comprehensive image analysis tool enables additional read-outs to quantify antitumoral activity of immune modulatory compounds. The latter can be used to identify possible biomarkers in the preclinical setting. Moreover, the translation and validation of these biomarker candidates in a clinical setting is self-evident as primary material needed for these types of analyses is easily accessible.
Citation Format: Daniel Bug, Eva Oswald, Anne Grote, Anne-Lise Peille, Gabriele Niedermann, Dorit Merhof, Friedrich Feuerhake, Julia B. Schüler. Humanized single mouse trial: A preclinical platform feasible for immune-oncology drug screening and translational biomarker development abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4815. doi:10.1158/1538-7445.AM2017-4815
Abstract
Experimental tumors raised in rodents represent an important preclinical tool to develop innovative anticancer compounds before clinical testing. Amongst others such models include solid ...tumors raised in syngeneic fully immunocompetent hosts and tumors spontaneously growing in genetically engineered mice (GEM) and derivate thereof. These model platforms have gained additional value since the manipulation of the immune system to fight cancer has led to tangible benefits for cancer patients. In the current study, we analyzed somatic mutation profiles from whole-exome sequencing (WES) data in a panel of 14 different mouse models covering 6 major cancer types. 4 models were GEM-derived, all other lines were developed by injection of established cell lines into the corresponding mouse strain. In parallel, these models were evaluated for their sensitivity towards checkpoint inhibitors (α-CTLA-4, α-PD-1 or α-PDL-1) in mono- or combined therapy with cytostatic and/or targeted agents.WES achieved an average-of-coverage of 165X in tumor models and normal DNA. A median mutation rate of 34 somatic mutations (m)/MB was detected, ranging from 7 m/MB (GEM derived NSCLC model KP) to 328 m/MB (syngeneic NSCLC line Lewis Lung) in exons. Mutation rates were markedly lower in GEM-derived models as in syngeneic lines (median of 9 vs 43 m/MB). This reflects very well the different underlying carcinogenic mechanism of these two types of models. The cross-comparison of tissue-transplants vs cell lines from GEM-derived model KP revealed that 75% of the mutations found in the primary KP could also be detected in the corresponding cell lines KP1 and KP4. Of note, the mutation count increased 1.3- (KP4) and 2.9-fold (KP1) during cell line establishment. Every model depicted a distinct profile against modulators of the immune system dividing the panel in responders and non-responders. In our hands no significant correlation could be determined between mutational load and sensitivity towards checkpoint inhibition in vivo. This might be related to the fact that the dataset was not broad enough and the number of models per entity was too small, rendering the subtype analysis within the panel not feasible. However, a strong tendency was observed when investigating the colon lines Colon26, CT26 and MC38 showing best response to the combination of PD-1+CTLA-4 inhibitors and in parallel the highest mutation rates (52, 64 and 59 m/MB, respectively) compared to non-responders B16-F10, CloudmanS91, 4T1 and KP1 (23 m/MB on average). Mouse models of cancer are a relevant tool for preclinical studies specifically for immuno-oncology. The molecular characterization of these models will help to optimize their use in drug discovery. They will support the development of innovative drugs and indentification of biomarkers to classify the patient cohort profiting the most from these new compounds.
Citation Format: Bruno Zeitouni, Cordula Tschuch, Jason M. Davis, Anne-Lise Peille, Yana Raeva, Manuel Landesfeind, Sheri Barnes, Julia B. Schüler. Whole-exome somatic mutation analysis of mouse cancer models and implications for preclinical immunomodulatory drug development abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1840. doi:10.1158/1538-7445.AM2017-1840
Abstract
Patient-derived tumor xenografts (PDX) have played a major role in the development of new cancer therapies and their strengths and weaknesses have gradually been elucidated. One major ...drawback of PDX is the lack of an immunological competent host. To overcome this hurdle we supplemented NSG/NOG mice with human hematopoietic stem cells (HSC) and subsequently examined growth characteristics of the non-small cell lung cancer (NSCLC) PDX model LXFA 923 in these mice. In parallel we monitored the presence of human and murine immune cells in different organs of the mouse.
HSC (2×106) cells were isolated from healthy donors and injected intravenously into sub-lethally irradiated NSG or NOG mice (n = 43 mice in 3 ind. exp.). After 8 weeks LXFA 923 was transplanted subcutaneously (s.c.) into the pretreated mice. Murine peripheral blood was examined by flow cytometry for common murine and human markers expressed on immune cells (hCD14, mCD14, hCD3, mCD3, hCD56, mCD56, hCD19) once weekly. At the end of the experiment tumors and organs were analyzed for human cancer (CD44, CD133, CDCP1, CD166, CD24) and immune cell markers (hCD14, hCD3, hCD56, hCD19) by flow cytometry. Tumors and organs were additionally histologically and immunohistochemically examined. Growth of the implanted tumors was monitored by caliper measurement. Mice bearing only the subcutaneous PDX or the HSC served as control groups.
Stable engraftment of human immune cells in immune-compromized mice was successfully achieved. Human immune cells expressing T-, B-, NK- and stem cell markers could be detected in different compartments (bone marrow, peripheral blood and spleen) of the tumor-bearing as well as non-tumor bearing mice. Furthermore, infiltrates of human monocytes (CD14+) as well as T cells (CD3+) could be detected in s.c. implanted tumor tissue. Implantation of LXFA 923 did not influence the proliferation of human immune cells in recipient mice. Growth behavior of the s.c. implanted PDX was not affected by the engraftment of HSC in the murine host. The histological architecture of LXFA 923 was similar when implanted s.c. in humanized or immunodeficient mice and it still closely resembles the patient donor material.
In conclusion, our investigations validate the analysis of PDX in mice engrafted with human immune cells, as it enables the interaction of tumor cells with human immune cells as well as with murine stroma to be investigated. This preclinical PDX based in vivo platform provides a further step to support the development of new drugs targeting the host immune response.
Citation Format: Eva Oswald, Kerstin Klingner, Dorothee Lenhard, Gabriele Niedermann, Julia B. Schüler. NSCLC PDX model for the evaluation of immuno-oncological treatment strategies. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5023. doi:10.1158/1538-7445.AM2015-5023
Abstract
Patient-derived xenografts (PDX) passaged in immunocompromised mice are a well-established system for preclinical efficacy testing of anti-cancer agents. Typically, PDXs are pre-screened ...either in vitro or via molecular analysis, and selected models are then tested in groups of 8 to 12 mice to confirm or evaluate the efficacy of a treatment relative to a vehicle control group. This approach results in highly reliable and reproducible efficacy data. However, it is often desirable to test large panels of tumor models in vivo in order to properly take genetic diversity into account. Such experiments can be cost-prohibitive in conventional study layouts, which results in fewer models being tested.
The Single Mouse Trial (SMT) format addresses the need for compound testing in larger, more diverse tumor populations. This format employs a single mouse per PDX model and treatment arm, thereby enabling the investigation of efficacy in substantially larger panels of PDX models. In order to better mirror inter-patient response diversity observed in the clinic, less emphasis is put on the statistical robustness of response data for individual models.
In the study presented here, six SoC drugs were tested in the SMT format in colorectal (cetuximab, oxaliplatin, irinotecan, 5-FU) and non-small-cell lung cancer (cetuximab, paclitaxel) PDXs, and results were compared to those obtained from standard format experiments with 5 - 10 mice per group. Dosing and schedules were adapted to clinical standards. Data for 19 CXF and 16 NSCLC models showed that in 77% of the cases, results obtained from the single mouse trial format were in line with results from standard efficacy tests. Furthermore, in 10% of the cases, efficacy was similar by trend between the two formats. Notable discrepancies were seen mainly in tumors exhibiting intermediate sensitivity.
Our findings indicate that for the drugs tested here, the risk of misjudging the efficacy in a given PDX model based on SMT is low. This risk could be further lowered by increasing group sizes to 3 mice. For the identification of biomarkers which requires accurate efficacy data, such an intermediate format between the SMT and the standard format might be advantageous.
Citation Format: Christina Gredy, Julia B. Schüler, Nina Zanella, Heinz-Herbert Fiebig, Thomas Metz. Single mouse trials, a concept using patient-derived tumor xenografts for large scale in vivo screens. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2890. doi:10.1158/1538-7445.AM2015-2890
Abstract
Introduction: Genetically engineered mouse models (GEMM) represent an attractive system for preclinical research since GEMM tumors develop in the presence of a competent immune system, ...thereby closely resembling the tumor microenvironment in patients. One example is the KP model (DuPage et al., 2009), which reflects the tumorigenesis of NSCLC in humans. KP tumors carry Kras and Trp53 mutations, comparable to subtypes of human NSCLC. Typical drawbacks of GEMM tumors include differences in their genetic make-up to human tumors and the very slow and heterogeneous tumor development, making the use of these models challenging for routine in vivo efficacy studies. The latter can be improved by grafting primary tumors on a genetically dissimilar member of the same strain, so-called allografting. Thus, the advantages of xenograft and GEMM models can be combined. Here, we described the establishment and characterization of allografts from the KP GEMM.
Material & Methods: KP tumors were excised from the lungs of KP mice and implanted subcutaneously (s.c.) into C57BL/6N female mice. When tumor growth was detected, passaging steps from animal to animal were performed. Furthermore, two cell lines derived from KP tumors, KP1 and KP4 (provided by Müller et al.), were injected s.c., the developing tumors excised and directly re-implanted as described above. The passages were analyzed molecularly, histologically, and via IHC (e.g. Ki67 proliferation marker) , and compared to the original in-situ tumors. A therapeutic study with Erlotinib and BEZ235 was performed on KP1 allografted mice.
Results: All investigated allografts showed similar histological patterns. Ki67 scores reflected the average time from implantation to passaging. The average passage times ranged from 19±3 days (KP4 cell line) to 26±6 days (KP1 cell line). Allografts from the original KP model showed a time to passaging of 23±5 days.
The mRNA expression of EpCAM (CD326) was on average 10-fold higher in KP and KP1 allografted tumors compared to normal C57BL/6 lung tissue, which could be confirmed by flow cytometry and cell-binding assay, determining >99% EPCAM positive cells in KP1 tumors. All allografts carried the Kras mutation and loss of function point mutations in Trp53.
The therapy study revealed no effect of Erlotinib on tumor growth, but a significant (p=0.0003) tumor load reduction in mice treated with BEZ235 (519 ± 244 mm³, n=5) compared to the control group (1678 ± 275 mm³, n=4) on day 28, respectively.
Conclusion: The generation of s.c. transplantable allografts from the KP GEMM was successfully conducted. The allograft models enable the use of immunocompetent mice in a feasible time frame and still resemble key aspects of human disease. Further studies will elucidate the interaction of the allografts with the host and compare sensitivity characteristics between GEMM and allograft of the same model.
Citation Format: Damaris Kukuk, Philipp Müller, Kerstin Klingner, Anne-Lise Peille, Alfred Zipelius, Julia B. Schüler. Establishment and characterization of allografts derived from a genetically engineered mouse model of non-small cell lung cancer (NSCLC). abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 100. doi:10.1158/1538-7445.AM2014-100
Abstract
Hematological malignancies account for about 10% of newly diagnosed cancers in the US, their heterogeneity and diverse biological characteristics present unique therapeutic challenges. In ...order to develop more promising therapeutic strategies, the establishment of functional and reproducible in vivo models is widely pursued. A panel of 18 hematological cell lines comprising different entities including leukemia (acute lymphoblastic AML; chronic myeloid CML; chronic lymphatic leukemia CLL), diffuse large B cell lymphoma (DLBCL), as well as multiple myeloma (MM) was evaluated for in vivo engraftment using different mouse strains and application routes.
18 different cell lines were inoculated into 4 different immuncompromized mouse strains (NOG; NSG; NOD/SCID; SCID/beige nude) in 35 independent experiments. Cells were injected intravenously (i.v.), intratibialy (i.t.) or intraperitonealy (i.p.). Tumor growth was monitored via a) determination of overall survival, b) fluorescence-based in vivo imaging (IVI, Bruker FX, using CF750 labeled anti-hu CD33, CD19, CD45) c) flow cytometry and d) histological and IHC examination.
Engraftment of hematological cell lines was clearly influenced by tumor entity, mouse strain and application route. The investigated AML cell lines engrafted in all three applied mouse strains (NOG; NSG; NOD/SCID) and injection routes. MOLM-13 infiltrated the bone marrow (BM) and the spleen (>90% infiltration, 21 days after tumor cell injection (=d21)), whereas MV4-11 cells disseminated mainly to the spleen (5% BM and 15% spleen infiltration, d21). In contrast THP-1 (80% BM and 2% spleen infiltration, d21) as well as NOMO-1 cells (30% BM and 3% spleen infiltration, d21) grew predominantly in the murine BM. MM cell lines (L363, RPMI8826, MM1R, MM1S) as well as B-CLL cell lines (Mec-1) failed to engraft reliably in NOD/SCID mice, irrespective of the application route. MM cells engrafted predominantly in the BM (20 - 60% depending on the cell line) of NSG mice when injected i.t.. i.v. injected MM cells did engraft in the BM but to a significantly lower extent than when injected i.t. (e.g. 20% vs. 2% BM infiltration of RPMI8226 cells, d35). Mec-1 cells infiltrated the BM and spleen of NSG and NOG mice reliably when injected i.v. and i.p. in the same individual (60% BM and 15% spleen infiltration, d28), whereas the same approach did not result in any engraftment when using SCID/beige nude mice. 3 out of 4 investigated DLBCL cell lines engrafted in vivo. Disseminated growth could be observed in NOG mice when injecting the cells i.v. or i.t. In all cases, tumors grew faster and infiltrated the BM more aggressively when injected I.t..
In summary, the engraftment of hematological cell lines strongly depends on the tumor microenvironment. The direct contact to the BM niche is a major survival benefit for the tumor cells as is the lack of NK cells in NOG and NSG mice.
Citation Format: Eva Oswald, Kerstin Klingner, Ralph Waesch, Katja Zirlik, Monika Engelhardt, Julia B. Schüler. Influence of tumor microenvironment on engraftment capacity of hematological cell lines in immunocompromized mice. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3926. doi:10.1158/1538-7445.AM2014-3926
Abstract
The generation of functional and reproducible in vivo models is a prerequisite for a better understanding of hematological malignancies and for the development of improved therapeutic ...strategies. Here, we report the establishment of transplantable PDX models of acute myeloid and lymphoid leukemia (AML, ALL) growing subcutaneously (s.c.) as well as in a disseminated fashion in immunocompromised mice.
Bone marrow (BM) and peripheral blood (PB) cells from 25 AML/ALL patients were injected intratibially into NSG or NOG mice (n = 1-9/patient) and in later passages also s.c. in nude mice (n = 1-3/model). Tumor growth was monitored via determination of overall survival, flow cytometry (verification of patient-derived leukemic clones by hCD34, hCD33, hCD38, hHLA-ABC, hCD3, hCD45, mCD45), and, where applicable, by caliper measurement. Engraftment is defined as ≥5% hCD45+ cells in the BM and/or ≥1% hCD45+ cells in PB. Transplantable models (higher or equal passage 2) were tested with the respective standard of care drugs (SoC) mainly Cytarabine and Dexamethasone.
Bone marrow engraftment in passage 1 was observed for 11 out of 25 leukemia models. Four of them could be established as disseminated models in serial passage. No engraftment was detected for nine models during the observation period of at least 95 days while observation for five models is still ongoing. The four established models were further characterized by fluorescence-in-situ-hybridization (FISH) to confirm patient origin. Treatment of mice bearing the models with the SoC drugs cytarabine and dexamethasone significantly prolonged overall survival. Antitumoral activity was consistent with clinical response of the donor patient. One T-ALL and two AMLs were additionally successfully propagated following s.c. injection in nude mice. They showed stable growth behavior and displayed a similar chemo-sensitivity pattern as their disseminated growing counterparts.
In conclusion, we have established a panel of transplantable PDX models of ALL and AML displaying stable and reproducible growth patterns suitable for in vivo efficacy studies. Our results confirm that PDX models of hematological malignancies replicate important clinical characteristics of the disease and are valuable tools for preclinical drug testing.
Citation Format: Eva Oswald, Kerstin Klingner, Benedikt Hammerich, Gabriele Greve, Dorothee Lenhard, Milena Pantic, Heinz-Herbert Fiebig, Michael Luebbert, Julia B. Schüler. Patient-derived models of human acute myeloid and lymphoid leukemia in immunocompromised mice for preclinical drug development. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4210. doi:10.1158/1538-7445.AM2015-4210
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