Bone metastasis occurs in up to 90% of men with advanced prostate cancer and leads to fractures, severe pain and therapy-resistance. Bone metastases induce a spectrum of types of bone lesions which can respond differently to therapy even within individual prostate cancer patients. Thus, the special environment of the bone makes the disease more complicated and incurable. A model in which bone lesions are reproducibly induced that mirrors the complexity seen in patients would be invaluable for pre-clinical testing of novel treatments. The microstructural changes in the femurs of mice implanted with PCSD1, a new patient-derived xenograft from a surgical prostate cancer bone metastasis specimen, were determined.Quantitative micro-computed tomography (micro-CT) and histological analyses were performed to evaluate the effects of direct injection of PCSD1 cells or media alone (Control) into the right femurs of Rag2-/-γc-/- male mice.Bone lesions formed only in femurs of mice injected with PCSD1 cells. Bone volume (BV) was significantly decreased at the proximal and distal ends of the femurs (p < 0.01) whereas BV (p < 0.05) and bone shaft diameter (p < 0.01) were significantly increased along the femur shaft.PCSD1 cells reproducibly induced bone loss leading to osteolytic lesions at the ends of the femur, and, in contrast, induced aberrant bone formation leading to osteoblastic lesions along the femur shaft. Therefore, the interaction of PCSD1 cells with different bone region-specific microenvironments specified the type of bone lesion. Our approach can be used to determine if different bone regions support more therapy resistant tumor growth, thus, requiring novel treatments.
A review of patients that were treated jointly by the plastic and the orthopedic surgery teams from 2011 to 2015 to evaluate the timing of definitive reconstruction of extremity defects was undertaken. The cohort consists of 81 patients, of which 4 presented with either a high-risk lesion or an advanced squamous cell carcinoma and of which the remaining 77 patients presented with different types of extremity sarcomas. In 58 cases, immediate-definitive reconstruction was performed, while in 23 cases the definitive procedure was delayed. Immediate definitive reconstruction, concurrent with ablative procedures, was offered whenever possible. In 16 cases, delayed reconstruction was due to diagnostic uncertainty (uncertain diagnosis per se or non-established negative margins). In 6 cases, delayed reconstruction was performed because of infection, in 1 case a bleeding disorder necessitated workup and in another, the intraoperative histology necessitated a plan-change, wound temporization and preparation of an arteriovenous fistula for microsurgical reconstruction. Indications for delayed reconstruction can be related to systemic conditions, donor or recipient site issues (infection, suspicion of margin-problems or tumor “not in continuity”). Although frozen sections have an overall diagnostic accuracy of 90%, changes in management were related to the inadequacy of frozen material, sampling errors resulting from heterogeneity or non-appositional growth of malignancy. A high rate (28%) of delayed reconstruction corresponds with new data reaffirming an old paradigm (radically excised margins “make a difference”), and with objectives dictated by customized, personalized, multimodal approaches. Wound temporization was offered whenever definitive reconstruction couldn’t be performed immediately. Limb sparing, with the utilization of temporary spacers or distractors, mega-prostheses, or trials of re-implanting formerly tumorous bone autografts, which have been sterilized to eliminate malignant cells – may all necessitate temporary coverage or staged repair. Authors’ clinical experience was evaluated in the context whether the rationale behind immediate versus delayed reconstruction was optimal.
Abstract Background: Acute myelogenous leukemia (AML) is a highly lethal disease with only 20% of 5 years survival. Progression to therapy resistant AML is driven by leukemia stem cells (LSC) harboring enhanced survival, dormancy and self-renewal capacity in supportive niches. Evidence indicates that in the development of AML deregulation of stem cells activity is as important as deregulation of the microenvironment. We have recently reported that inflammatory changes in the aging bone marrow (BM) niche via aberrant RNA editing and splicing may predispose patients to leukemic transformation. However, little is known about the functional effects of niche driven aberrant RNA processing in the AML pathogenesis, as well as of role of aberrant RNA editing and splicing in human aged stem cell fate, dormancy and regeneration in the context of microenvironment. Here we investigate: 1/ the LSC impacts to the BM niche and 2/ changes in the activity of BM niche cells contribute to AML pathogenesis. Methods: BM CD34- cell from normal donors and AML patients were used for the development of the primary human stromal monolayers : young (y-BM, n=3); old (a-BM, n=3) and AML (n=3). Human CD34+ cells were selected from AML primary samples (n=6). As a control, CD34+ cells from cord blood (CB, n=5), y-BM (n=3) or a-BM (n=3) were used for the co-culture experiments and then plated in survival and self-renewal assays. Results: AML- and normal BM-derived stroma differ in their ability to support HSC and LSC: LSC (n=6) were capable to self-renew after 9 weeks of co-culture with both normal and AML stroma, while cord blood HSC (n=5) lost their self-renewal potential after only 2 weeks of co-culture with the AML stroma. Aged BM also impaired survival and self-renewal of cord blood HSC (n=3) in stromal co-culture models. In similar experiments HSC from a-BM (n=3) demonstrated significantly higher survival and self-renewal capacity then co-cultured with y-BM stroma (n=3) compared to a-BM stroma (n=3). Conditioned media (CM) both from-, or co-culture with-, old or AML stroma impaired HSC survival and self-renewal. Notably, co-culture conditions resulted in a greater reduction in survival and self- renewal capacity, suggesting that cell-cell contact or unstable secreted factors exacerbate the effects. Cord blood HSC were also co-cultured with AML-derived stroma or CM , and this severely impaired their survival and self-renewal. Moreover, pre-treatment of the HS-5 cells with CM from a-BM or AML stroma for 4 weeks prior to co-culture experiments led to significant inhibition of the cord blood HSC (n=3) survival and self-renewal. Conclusions: Together these data indicate that microenvironmental cues play a key role in regulating normal HSC versus LSC survival and maintenance, and leukemic and aged stroma exibit severely compromised ability to maintain normal HSCs, but effectively support LSCs. Targeting this pathological interplay could represent a novel avenue for treatment of AML. Citation Format: Larisa Balaian, Leslie Crews, Anna Kulidjian, Edward Ball, Catriona Jamieson. Differential survival and self-renewal of LSC and HSC in aged and leukemic niches. [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 4865. doi:10.1158/1538-7445.AM2014-4865
Abstract Introduction: Human bone marrow aging is typified by decreased cellularity, stem cell exhaustion and myeloid lineage bias that may set the stage for development of myeloid malignancies. Secondary AML (sAML) is a malignancy that has been associated with alterations in RNA processing genes and currently has few effective treatment options available. A central goal of future therapeutic strategies is to prevent disease relapse and therapeutic resistance by selectively targeting unique gene products that are essential to LSC but not normal HSC function. Therefore, we established whole gene, long non-coding RNA (lncRNA), splice isoform, and RNA editing signatures of benign versus malignant bone marrow progenitor cell aging, and evaluated the therapeutic efficacy of splicing-targeted agents in pre-clinical humanized in vitro and in vivo model systems. Methods: Whole transcriptome sequencing (RNA-Seq) was performed on FACS-purified hematopoietic stem (CD34+CD38-Lin-) and progenitor cells (CD34+CD38+Lin-) from aged (average age = 65.9 ± 6.8 years old) versus young (average age = 25.8 ± 3.0 years old) adult healthy bone marrow samples, and in leukemia stem cells (LSC) from patients with sAML (average age = 71.4 ± 7.9 years old). Comparative gene set enrichment analyses (GSEA), splice isoform, lncRNA, and RNA editing profiles were identified for normal and malignant progenitor cell aging. Then, we evaluated the spliceosome modulatory agent 17S-FD-895 in splicing reporter activity, PCR, and functional in vitro hematopoietic progenitor and in vivo LSC primagraft assays. Results: Disruption of pre-mRNA splicing activity has recently been implicated as a therapeutic vulnerability in some types of cancer. Comparative whole transcriptome RNA sequencing (RNA-seq) analyses revealed pre-mRNA splicing factor gene expression was significantly disrupted in human AML LSC compared with age-matched normal progenitors. Comparative splice isoform RNA-seq and qRT-PCR validation revealed recurrent intron retention and exon skipping in expressed transcripts, such as PTK2B and several protein phosphatase gene products. Notably, transcription factor profiling of AML LSC demonstrated downregulation of key tumor suppressor genes, such as IRF8 and TP53. We then investigated the LSC inhibitory efficacy of a stable and potent splicing modulatory agent, 17S-FD-895, in humanized stromal co-culture and AML LSC primagraft assays. Pharmacological spliceosome modulation disrupted AML LSC maintenance in vivo by altering splicing of stem cell survival and AML-associated transcripts at doses that spared normal hematopoietic progenitors. Conclusions: Detection and targeted modulation of aberrant RNA processing provides an innovative strategy for AML LSC eradication with implications for treatment of a variety of human malignancies and other age-related disorders. Citation Format: Leslie A. Crews, Larisa Balaian, Heather S. Leu, Nathaniel P. Delos Santos, Angela C. Court, Anil Sadarangani, Maria A. Zipeto, James J. La Clair, Reymundo Villa, Sheldon R. Morris, Rainer Storb, Anna Kulidjian, Edward D. Ball, Michael D. Burkart, Catriona H.M. Jamieson. RNA processing signatures of normal versus malignant progenitor cell aging predict leukemia stem cell sensitivity to RNA splicing modulation. [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 915.
Abstract Background: Upon diagnosis of castration-resistant prostate cancer (CRPC), 84% of patients present with metastases and their survival rate is 28% despite improvements in treatment options. PCSD1, a patient-derived xenograft from a prostate cancer bone metastasis replicated the donor patient's CRPC and was resistant to the anti-androgen drug, bicalutamide, in the bone niche. To further understand the mechanisms of androgen deprivation therapy (ADT) resistance, we treated PCSD1 and PCSD13, a novel patient derived xenograft model from a small cell prostate cancer bone metastasis, with the more potent anti-androgen, Enzalutamide. Methods: PDX cells were implanted into male Rag2−/−γc−/− mice intra-femorally (IF) or subcutaneously (SC). Mice with established tumors were treated o.g. for 4-5 weeks with Enzalutamide (10mg/kg/day) or Vehicle control. Tumor growth was measured bi-weekly with calipers and in vivo bioluminescence (IVIS). Tumors were analyzed using FACS, RNASeq, whole exome sequencing (WES) and Oncoscan. Results: PCSD1 and PCSD13 tumors were resistant to enzalutamide when implanted in the femur bone but not sub-cutaneously. When the enzalutamide-resistant tumor cells from the bone were re-injected sub-cutaneously they retained their enzalutamide resistance in contrast to treatment-naïve PCSD1 SC tumors. Transcriptomic analysis revealed significant modulation of WNT5A, EMT and neuronal programs. Single cell analyses revealed heterogeneous sub-populations that behave as cancer stem cells in 3D organoid cultures. Genome-wide CNV analysis of patient bone metastasis samples from the same patient obtained 10 months apart and his xenografts revealed extensive CNVs which were highly conserved between the patient samples and xenografts. Conclusions: Our PDX models demonstrated that the bone niche promotes tumor growth and malignant progression even when treated with the potent anti-androgen, enzalutamide. These PDX models of bone metastatic prostate cancer were resistant to enzalutamide in the femur bone (IF) but sensitive in the sub-cutaneous environment (SC). Enzalutamide resistance in the bone produced a permanent change in the tumor cells that was retained when re-injected in the sub-cutaneous location. This elucidates the importance of utilizing in vivo patient derived models to gain a better understanding of why the bone niche supports tumor growth. Citation Format: Christina A.M. Jamieson, Michelle T. Muldong, Sanghee Lee, Christina N. Wu, Danielle N. Burner, Theresa R. Mendoza, Catalina Arreola, Abril Zuniga, Nicholas A. Cacalano, Catriona H. Jamieson, Christopher J. Kane, Anna A. Kulidjian. Enzalutamide treatment of patient derived bone metastatic prostate cancer xenograft models implanted in the bone resulted in durable progression to castration resistant prostate cancer (CRPC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6109.
Prostate cancer bone metastasis occurs in 50-90% of men with advanced disease for which there is no cure. Bone metastasis leads to debilitating fractures and severe bone pain. It is associated with therapy resistance and rapid decline. Androgen deprivation therapy (ADT) is standard of care for advanced prostate cancer, however, bone metastatic prostate cancer (PCa) often becomes resistant to ADT. There are few pre-clinical models to understand the interaction between the bone microenvironment and prostate cancer. Here we report the castrate resistant growth in the bone niche of PCSD1, a patient-derived intra-femoral xenograft model of prostate bone metastatic cancer treated with the anti-androgen, bicalutamide. PCSD1 bone-niche model was derived from a human prostate cancer femoral metastasis resected during hemiarthroplasty and serially transplanted into Rag2 −/− ;γ c −/− mice intra-femorally (IF) or sub-cutaneously (SC). At 5 weeks post-transplantation mice received bicalutamide or vehicle control for 18 days. Tumor growth of PCSD1 was measured with calipers. PSA expression in PCSD1 xenograft tumors was determined using quantitative RT-PCR and immunohistochemistry. Expression of AR and PSMA, were also determined with qPCR. PCSD1 xenograft tumor growth capacity was 24 fold greater in the bone (intra-femoral, IF) than in the soft tissue (sub-cutaneous, SC) microenvironment. Treatment with the anti-androgen, bicalutamide, inhibited tumor growth in the sub-cutaneous transplantation site. However, bicalutamide was ineffective in suppressing PCSD1 tumor growth in the bone-niche. Nevertheless, bicalutamide treatment of intra-femoral tumors significantly reduced PSA expression (p < =0.008) and increased AR (p < =0.032) relative to control. PCSD1 tumors were castrate resistant when growing in the bone-niche compared to soft tissue. Bicalutamide had little effect on reducing tumor burden in the bone yet still decreased tumor PSA expression and increased AR expression, thus, this model closely recapitulated castrate-resistant, human prostate cancer bone metastatic disease. PCSD1 is a new primary prostate cancer bone metastasis-derived xenograft model to study bone metastatic disease and for pre-clinical drug development of novel therapies for inhibiting therapy resistant prostate cancer growth in the bone-niche.
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