The Role of Human Circulating Hematopoietic Stem and Progenitor Cells (CHSPCs) in Promoting Tumor Growth, Angiogenesis and Vascular Repair March 3rd, 2011 Jamie Case, PhD Assistant Research Professor of Pediatrics Indiana University School Of Medicine Director, Angiogenesis, Endothelial & Pro-Angiogenic Cell Core Indiana University Simon Cancer Center

EPCs And CPCs In Human Peripheral Blood Angiogenesis is crucial for tumor growth, and novel anti-angiogenic therapies are currently being tested as a means of slowing or preventing growth of tumors. In the past decade, important insights on the role of BM derived cells in adult neovascularization have been gained. In particular, EPCs and CPCs have been suggested to home to sites of neovascularization and neoendothelialization and differentiate into ECs in situ. EPCs and CPCs are being used as a biomarker for tumor angiogenesis as they are reported to function in vasculogenesis and angiogenesis. EPCs and CPCs have been proposed as a possible target to restrict vessel growth in tumor pathology. We know that Angio….. In the past decade, important insights….. Have been gained. In particular…. EPCc and CPCs are Therefore…..

EPCs And CPCs As Biomarkers The blood concentrations of circulating endothelial and progenitor cells are elevated in cancer patients. Little question that EPCs or circulating angiogenic cells are important biomarkers of disease and for measuring the impact of anti-angiogenic treatments of cancer. Enumerating these cells in blood cultures has yielded correlations between the number of cultured cells and severity of disease. Culturing blood is slow and costly and may not accurately represent the blood cell concentration. Various flow cytometry strategies have been employed to measure cell concentration. The…. Therefore, there is….. In fact…. However, Therefore, various …..

Poly-Chromatic Flow Cytometry Developed a novel protocol for the phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood. Highly reproducible Intended for longitudinal studies. Eliminates ambiguous data interpretation. Allows for cross-study comparisons. Cost and time effective Uses CD34, AC133, CD45, CD14 and CD31 to enumerate two biomarker populations. Simple protocol can be completed in 2-2.5 hours.

PFC Identifies Two Distinct Cell Populations Pro-Circulating Progenitor Cells (CPCs) (CD31+CD45dimCD34brightAC133+) Non-Circulating Progenitor Cells (Non-CPCs) (CD31+CD45dimCD34brightAC133-) Using PFC, 2 populations were identified..

Application Of PFC: CPC Analysis Applied PFC in which monocytes are excluded based on forward and side scatter (and confirmed with CD14 backgating because they auto-fluoresce and overlap the CPC population) as well as utilizing a dump channel with glyA and ViViD. CD34brightAC133+CD45dimCD31+

CPCs in a normal PB sample range from 0.05-0.15% of the total MNCs. In cancer patients the CPCs range from 1-3%. The frequency of CPCs in normal PB ranges from 0.05-0.15%, and in cancer patients, this range increases to around 1-2%.

Interestingly, many groups gate on the CPCs in a CD34/45 plot Interestingly, many groups gate on the CPCs in a CD34/45 plot. Correct use of FMOs show us that this is actually a heterogeneous population of cells, of which only a fraction are AC133+. The remaining fraction is what we have dubbed the non-CPCs are AC133 negative.

CPC/AC133+/- Populations CD34 This slide just shows you the 2 fractions when using the appropriate FMO gating. AC133

Why Are These Subtle Differences In Phenotype Important? Metastatic Breast Cancer Untreated Sunitinib Taxol So why are these subtle differences in phenotype important? This is an example of a patient with metastatic breast cancer undergoing a series of treatments. Here we’re tracking the progenitor population which you can see is inhibited by sunitinib and then partially restored after the addition of paclitaxel to the treatment regime.

Metastatic Breast Cancer Untreated Sunitinib Taxol However, when we take a closer look at the phenotype of this population we see that the true CPCs, those expressing 133 are not restored by treatment with paclitaxel. Instead it is the Non-CPC fraction that is actually being mobilized. So in seeing this we wanted to know if these 2 populations were functionally different and their role/function in cancer and cancer progression.

Are these 2 populations functionally different and what is their role/function? The number of CPCs in PB has been shown to clearly correlate with extent of tumor progression and CVD risk.

Rationale and Objective To investigate whether CPCs play a role in tumor progression…. Develop xenograft models to specifically study CPC function in tumor progression. Develop a more clinically relevant xenograft model to test therapies that target CPCs. Therefore,

Do Isolated CPCs Augment Tumor Growth? Non-CPCs CD34+ PBS HUMAN Melanoma (C32) 10-12 days NOD/SCID So, as the number of CPCs in PB has been shown to clearly correlate with extent of tumor progression and CVD risk, yet the functional identity of the cells is unknown, we asked the question… Monitor Tumor Growth

Tail Vein Injection Of CPCs Increases Tumor Growth Expt II:- 0.5x105 CPCs/mouse Expt I:- 1x105 CPCs/mouse * * while the non-CPCs performed no better than the vehicle control (PBS), the CPCs significantly increased the tumor size and weight. *p<0.001 CPCs vs CD34+ or PBS

Increased Vascular Density in Tumors Derived from Mice Injected with CPCs

Pro-CPC vs Non-CPC Antigen Expression Profile Data suggests that the CPCs (Black bars) are committed myeloid progenitors whereas the non-CPC population is a heterogeneous mixture of lymphoid progenitors. So when we examine the ratios of CPCs to non-CPCs as a biomarker of disease we get some pretty striking trends

Ratio Of CPCs Versus non-CPCs In Disease In normals: ratio is 1.2-2 Cancer: ratio is 2.5+ Diabetes: ratio is <1 PAD: ratio is <1 Breast Cancer before Sunitinib: ratio >2.5 After Sunitinib: ratio is 1-1.5 After coupling with Taxol: ratio <1 Ratios can be helpful to normalize wide population variances seen in absolute numbers that can mask significant trends just as the CD4/8 ratio is used to monitor HIV progression. Actually in the normal range we see it is very tight as compared to the CD4/8 ratio.

Pro-CPC:Non-CPC Ratio In PVD And Breast Cancer *** = 0.0001 ** = 0.01 * = 0.05 *p=0.05 **p=0.01 ***p=0.0001

Interaction between the CPCs and the ECFCs as we have shown tumors were increased following CPC injection. CPCs home to the junctions and provide increased stability and prevent apoptosis compared to tubes formed in matrigel with ECFCs alone.

CPC ELISA for Angiogenic Growth Factors Pro-CPCs were shown to have elevated levels of IL-6, MIP-1a, MCP-1, VEGF, GM-CSF and G-CSF in condition media compared to controls. IL-2, IL-4, FGF-b, IFN-g, TNF-a, PDGF-b, b-NGF, SCF, SDF-1a were not elevated. Controls are ECFCs alone C32 alone

CPC Conclusions Pro-CPCs are enriched for hematopoietic stem cells of the myeloid lineage and hematopoietic cells that augment tumor growth in vivo. (Estes et al., Cytometry A, 77A(9):831-839, 2010) At present, the functional consequence of CPC elevation in cancer patients in not known. With the advent of PFC, it is now possible to isolate and rigorously characterize both the subsets of circulating angiogenic cells, including the Pro- and Non-CPCs. By gaining a detailed understanding of CPC function, the validity of the CPC as a new therapeutic target can be established. CPCs… At present…. However, with the …. So by gaining..

CPC Conclusions Cont’d Disparate angiogenic potential of the two subsets observed within the putative CPC population led us to hypothesize that these two progenitor fractions regulate a balance between vascular dysfunction and tumor angiogenesis. Observed disruptions in their relative frequencies may signal impaired or enhanced angiogenic function. Possible that CPCs represent a newly discovered cellular target that could be therapeutically modulated in the quest to kill cancer. CPCs may represent a powerful biomarker to monitor disease progression and treatment response. The disparate…… Therefore, it is very possible that… Thus it is possible… In addition, CPCs….

Acknowledgments Case Lab Pollok Lab Tony Sinn Julie Mund Haiyan Wang Barbara Bailey Jayne Silver Julie Mund Matt DiStasi EPCs are thought to circulate in PB, home to sites of new blood vessel formation and facilitate either arteriogenesis or angiogenesis by direct integration into the emerging endothelium or via paracrine stimulation of existing vessel wall derived cells. Therefore, EPCs or circulating angiogenic cells have been proposed as a possible target to restrict vessel growth in tumor pathology. Recently, the clinical utility of anti-angiogenic therapy has been established. Increased survival rates in phase III clinical trials were observed with the addition of vascular endothelial growth factor (VEGF) specific antibody (Avastin) to contemporary therapy for breast, lung, colon and rectal cancer patients. Furthermore, data has emerged from trials of tyrosine kinase inhibitors (Sutent and Nexavar) whose inhibitory spectra include VEGF-receptor kinases showing similar results. The blood concentrations of circulating endothelial and progenitor cells are elevated in cancer patients, therefore, there is little question that EPCs or circulating angiogenic cells are important biomarkers of disease and for measuring the impact of anti-angiogenic treatments of cancer. Hematopoietic cells and myeloid progenitor cells are known to participate in angiogenesis and are involved in crucial processes during tumor development. Developing tumors build intra-tumoral blood vessels by co-opting neighboring, pre-existing blood vessels, which are activated and expand within the growing tumor mass as a result of the brisk proliferation of endothelial cells (ECs) under the influence of tumor-derived pro-angiogenic signals. Several tumor-secreted factors can recruit hematopoietic cells, which, in turn, stimulate angiogenesis by secreting pro-angiogenic mediators. Once recruited to the tumor microenvironment, hematopoietic cells might exert a paracrine effect on the angiogenic process via a number of different means: (1) direct stimulation of ECs leading to EC proliferation and their acquisition of a migratory and invasive phenotype; (2) release of both soluble and matrix-bound pro-angiogenic factors; (3) assistance of ECs during cell migration and vessel morphogenesis, and (4) suppression of anti-tumor immunity. Moreover, several hematopoietic cell subsets of the myeloid lineage have been shown to have pro-angiogenic activity in tumors.