Kenneth C. Valkenburg

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation.

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Drug discovery in prostate cancer mouse models

Introduction: The mouse is an important, though imperfect, organism with which to model human disease and to discover and test novel drugs in a preclinical setting. Many experimental strategies have been used to discover new biological and molecular targets in the mouse, with the hopes of translating these discoveries into novel drugs to treat prostate cancer in humans. Modeling prostate cancer in the mouse, however, has been challenging, and often drugs that work in mice have failed in human trials. Areas covered: The authors discuss the similarities and differences between mice and men; the types of mouse models that exist to model prostate cancer; practical questions one must ask when using a mouse as a model; and potential reasons that drugs do not often translate to humans. They also discuss the current value in using mouse models for drug discovery to treat prostate cancer and what needs are still unmet in field. Expert opinion: With proper planning and following practical guidelines by the researcher, the mouse is a powerful experimental tool. The field lacks genetically engineered metastatic models, and xenograft models do not allow for the study of the immune system during the metastatic process. There remain several important limitations to discovering and testing novel drugs in mice for eventual human use, but these can often be overcome. Overall, mouse modeling is an essential part of prostate cancer research and drug discovery. Emerging technologies and better and ever-increasing forms of communication are moving the field in a hopeful direction.

A simple selection-free method for detecting disseminated tumor cells (DTCs) in murine bone marrow

Bone metastasis is a lethal and incurable disease. It is the result of the dissemination of cancer cells to the bone marrow. Due to the difficulty in sampling and detection, few techniques exist to efficiently and consistently detect and quantify disseminated tumor cells (DTCs) in the bone marrow of cancer patients. Because mouse models represent a crucial tool with which to study cancer metastasis, we developed a novel method for the simple selection-free detection and quantification of bone marrow DTCs in mice. We have used this protocol to detect human and murine DTCs in xenograft, syngeneic, and genetically engineered mouse models. We are able to detect and quantify bone marrow DTCs in mice that do not have overt bone metastasis. This protocol is amenable not only for detection and quantification purposes but also to study the expression of markers of numerous biological processes or tissue-specificity.

Deletion of tumor suppressors adenomatous polyposis coli and Smad4 in murine luminal epithelial cells causes invasive prostate cancer and loss of androgen receptor expression

Prostate cancer is the most diagnosed non-skin cancer in the US and kills approximately 27,000 men per year in the US. Additional genetic mouse models are needed that recapitulate the heterogeneous nature of human prostate cancer. The Wnt/beta-catenin signaling pathway is important for human prostate tumorigenesis and metastasis, and also drives tumorigenesis in mouse models. Loss of Smad4 has also been found in human prostate cancer and drives tumorigenesis and metastasis when coupled with other genetic aberrations in mouse models. In this work, we concurrently deleted Smad4 and the tumor suppressor and endogenous Wnt/beta-catenin inhibitor adenomatous polyposis coli (Apc) in luminal prostate cells in mice. This double conditional knockout model produced invasive castration-resistant prostate carcinoma with no evidence of metastasis. We observed mixed differentiation phenotypes, including basaloid and squamous differentiation. Interestingly, tumor cells in this model commonly lose androgen receptor expression. In addition, tumors disappear in these mice during androgen cycling (castration followed by testosterone reintroduction). These mice model non-metastatic castration resistant prostate cancer and should provide novel information for tumors that have genetic aberrations in the Wnt pathway or Smad4.Prostate cancer is the most diagnosed non-skin cancer in the US and kills approximately 27,000 men per year in the US. Additional genetic mouse models are needed that recapitulate the heterogeneous nature of human prostate cancer. The Wnt/beta-catenin signaling pathway is important for human prostate tumorigenesis and metastasis, and also drives tumorigenesis in mouse models. Loss of Smad4 has also been found in human prostate cancer and drives tumorigenesis and metastasis when coupled with other genetic aberrations in mouse models. In this work, we concurrently deleted Smad4 and the tumor suppressor and endogenous Wnt/beta-catenin inhibitor adenomatous polyposis coli (Apc) in luminal prostate cells in mice. This double conditional knockout model produced invasive castration-resistant prostate carcinoma with no evidence of metastasis. We observed mixed differentiation phenotypes, including basaloid and squamous differentiation. Interestingly, tumor cells in this model commonly lose androgen receptor expression. In addition, tumors disappear in these mice during androgen cycling (castration followed by testosterone reintroduction). These mice model non-metastatic castration resistant prostate cancer and should provide novel information for tumors that have genetic aberrations in the Wnt pathway or Smad4.

Murine Prostate Micro-dissection and Surgical Castration.

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

Abstract 4109: Unbiased detection of disseminated tumor cells in murine bone marrow

Approximately 20-30% of prostate cancer patients develop disease recurrence and metastasis years after initial therapy. This is thought to be largely due to the presence of growth-arrested and chemoresistant disseminated tumor cells (DTCs) in secondary sites, such as bone. Bone metastasis is found in 90-100% of prostate cancer patients who succumb to the disease. There are still many gaps in knowledge about the biological mechanisms by which DTCs home to bone, resist chemotherapy, become dormant, and escape dormancy to grow into clinical metastases. As such, it is important to be able to detect, quantify, and study bone marrow DTCs. In particular, it must be possible to do this in metastatic cancer mouse models, which are critical to study the process of tumor dissemination. DTC detection techniques currently exist, usually as either a positive selection or negative selection methodology. Positive selection techniques use markers or cell size to isolate and purify tumor cells out of the bone marrow. Positive selection markers are generally epithelial-specific, such as EpCam, E-cadherin, or Cytokeratin, and therefore may miss cells that lose epithelial marker expression and may gain mesenchymal markers. DTCs can also be as small as or smaller than white blood cells, meaning that positive selection based on size may miss some DTCs. Negative selection enriches for DTCs by removing blood and bone marrow cells from the population, usually using cell-specific markers. A popular strategy is CD45-based depletion, which removes white blood cells, and theoretically leaves behind DTCs. In our hands, this strategy causes loss of DTCs in the depletion process. To capture these heterogeneous and rare DTCs, we have developed a strategy to detect DTCs in murine bone marrow in an un-biased manner. The procedure entails removal of the bone marrow via centrifugation from the long bones (femur and tibia) of mice that have been injected with cancer cells (the injection site may vary depending on the experimental setup). The bone marrow then undergoes red blood cell lysis, and further centrifugation. The white blood cells are then counted, and the bone marrow is spread onto glass slides. The cells on the slide are fixed, permeabilized, and stained (immunofluorescence and RNA fluorescent in situ hybridization can be used). The staining can include any type of marker, including epithelial, mesenchymal, disease-specific, species-specific, or other biologically interesting markers, such as cell cycle markers. The unbiased nature of this procedure is based on the lack of positive or negative selection based on cell size or protein expression. Some DTC loss is noted in this protocol, due to the centrifugation and staining steps, but the cell population on the slide should include all DTC types. Notably, this protocol can be used to detect human or mouse cells in the mouse bone marrow and can thus be used in immune-compromise and immune-competent mouse models of metastasis. Citation Format: Kenneth C. Valkenburg, James R. Hernandez, Sarah R. Amend, James E. Verdone, Michael A. Gorin, Kenneth J. Pienta. Unbiased detection of disseminated tumor cells in murine bone marrow. [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 4109.

Abstract 365: Mobilizing prostate cancer cells from the endosteal niche by targeting the SDF-1/CXCR4 axis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Metastatic prostate cancer cells preferentially home to bone, and skeletal metastases are present in approximately 90% of patients upon autopsy. Our lab was the first to show that prostate cancer cells compete with hematopoietic stem cells (HSCs) for space within the endosteal niche. The HSC niche is located on the surface of endosteal osteoblasts, where HSCs are kept in a slow-cycling, protective state. The niche also provides prostate cancer cells with a chemoprotective environment in which they can remain dormant for an extended period of time, even decades. C-X-C chemokine receptor 4 (CXCR4) is required for HSC recruitment to the HSC niche. Osteoblasts secrete stromal cell derived factor 1 (SDF-1, also known as CXCL12), the ligand for CXCR4, which attracts HSCs to the niche via a chemotactic gradient. CXCR4 is also expressed by disseminated prostate cancer cells and is required for prostate cancer cells homing to bone. HSCs can be mobilized from the niche into the bloodstream physiologically via infection or stress and pharmacologically via proteins like granulocyte-colony stimulating factor (G-CSF) or small molecule inhibitors like AMD3100 (plerixafor). AMD3100, which antagonizes CXCR4, is capable of mobilizing both HSCs and prostate cancer cells from the niche. When combined with AMD3100, docetaxel (the standard of care for prostate cancer) is more effective at killing tumor cells than when given as a single agent. This indicates that mobilization of disseminated tumor cells (DTCs) from the endosteal niche by targeting the SDF-1/CXCR4 axis might cause these cells to be more sensitive to chemotherapy in patients. This strategy would theoretically kill DTCs, which, when they become metastatic lesions, lead to most of the morbidity and mortality that affects prostate cancer patients. We are engaged in a Phase 0 clinical trial in metastatic prostate cancer patients to test this strategy in human patients. To complement the human trial, we are testing several CXCR4 inhibitors combined with G-CSF and docetaxel. We are able to detect CTCs in human patients, as well as CTCs and DTCs in mice via several techniques, and will use this technology as a readout for the clinical trial, as well as the preclinical studies. Citation Format: Kenneth C. Valkenburg, Kenneth J. Pienta. Mobilizing prostate cancer cells from the endosteal niche by targeting the SDF-1/CXCR4 axis. [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 365. doi:10.1158/1538-7445.AM2015-365