Mesenchymal Plasticity Regulated by Prrx1 Drives Aggressive Pancreatic Cancer Biology.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibroblast-rich desmoplastic stroma. Cancer-associated fibroblasts (CAFs) have been shown to display a high degree of interconvertible states including quiescent, inflammatory, and myofibroblastic phenotypes; however, the mechanisms by which this plasticity is achieved are poorly understood. Here, we aim to elucidate the role of CAF plasticity and its impact on PDAC biology. METHODS: To investigate the role of mesenchymal plasticity in PDAC progression, we generated a PDAC mouse model in which CAF plasticity is modulated by genetic depletion of the transcription factor Prrx1. Primary pancreatic fibroblasts from this mouse model were further characterized by functional in vitro assays. To characterize the impact of CAFs on tumor differentiation and response to chemotherapy, various coculture experiments were performed. In vivo, tumors were characterized by morphology, extracellular matrix composition, and tumor dissemination and metastasis. RESULTS: Our in vivo findings showed that Prrx1-deficient CAFs remain constitutively activated. Importantly, this CAF phenotype determines tumor differentiation and disrupts systemic tumor dissemination. Mechanistically, coculture experiments of tumor organoids and CAFs showed that CAFs shape the epithelial-to-mesenchymal phenotype and confer gemcitabine resistance of PDAC cells induced by CAF-derived hepatocyte growth factor. Furthermore, gene expression analysis showed that patients with pancreatic cancer with high stromal expression of Prrx1 display the squamous, most aggressive, subtype of PDAC. CONCLUSIONS: Here, we define that the Prrx1 transcription factor is critical for tuning CAF activation, allowing a dynamic switch between a dormant and an activated state. This work shows that Prrx1-mediated CAF plasticity has significant impact on PDAC biology and therapeutic resistance.

Metastasis and chemoresistance in CD133 expressing pancreatic cancer cells are dependent on their lipid raft integrity.

Metabolic rewiring is an integral part of tumor growth. Among metabolic pathways, the Mevalonic-Acid-Pathway (MVAP) plays a key role in maintaining membrane architecture through cholesterol synthesis, thereby affecting invasiveness. In the current study, we show for the first time that CD133Hi pancreatic tumor initiating cells (TIC) have increased expression of MVAP enzymes, cholesterol-content and Caveolin expression. Further, we show that CD133 in these cells is localized in the lipid-rafts (characterized by Cav-1-cholesterol association). Disruption of lipid-rafts by either depleting Cav-1 or by inhibiting MVAP by lovastatin decreased metastatic-potential and chemoresistance in CD133Hi cells while not affecting the CD133lo cells. Additionally, disruption of lipid-raft results in deregulation of FAK-signaling, decreasing invasiveness in pancreatic-TICs. Furthermore, this also inhibits ABC-transporter activity resulting in sensitizing TICs to standard chemotherapeutic agents. Repurposing existing drugs for new clinical applications is one of the safest and least resource intensive approaches to improve therapeutic options. In this context, our study is extremely timely as it shows that targeting lipid-rafts with statins can sensitize the normally resistant pancreatic TICHi-cells to standard chemotherapy and decrease metastasis, thereby defining a novel strategy for targeting the TICHi-PDAC.

GRP78-mediated antioxidant response and ABC transporter activity confers chemoresistance to pancreatic cancer cells.

Chemoresistance is a major therapeutic challenge that plays a role in the poor statistical outcomes in pancreatic cancer. Unfolded protein response (UPR) is one of the homeostasis mechanisms in cancer cells that have been correlated with chemoresistance in a number of cancers including pancreatic cancer. In this study, we show that modulating glucose regulatory protein 78 (GRP78), the master regulator of the UPR, can have a profound effect on multiple pathways that mediate chemoresistance. Our study showed for the first time that silencing GRP78 can diminish efflux activity of ATP-binding cassette (ABC) transporters, and it can decrease the antioxidant response resulting in an accumulation of reactive oxygen species (ROS). We also show that these effects can be mediated by the activity of specificity protein 1 (SP1), a transcription factor overexpressed in pancreatic cancer. Thus, inhibition of SP1 negatively affects the UPR, deregulates the antioxidant response of NRF2, as well as ABC transporter activity by inhibiting GRP78-mediated ER homeostasis. Sp1 and NRF2 have been classified as nononcogene addiction genes and thus are imperative to understanding the molecular mechanism of resistance. These finding have huge clinical relevance as both Sp1 and GRP78 are overexpressed in pancreatic cancer patients and increased expression of these proteins is indicative of poor prognosis. Understanding how these proteins may regulate chemoresistance phenotype of this aggressive cancer may pave the way for development of efficacious therapy for this devastating disease.

NFκB-Mediated Invasiveness in CD133+ Pancreatic TICs Is Regulated by Autocrine and Paracrine Activation of IL1 Signaling.

Tumor-initiating cells (TIC) have been implicated in pancreatic tumor initiation, progression, and metastasis. Among different markers that define this cell population within the tumor, the CD133+ cancer stem cell (CSC) population has reliably been described in these processes. CD133 expression has also been shown to functionally promote metastasis through NF-κB activation in this population, but the mechanism is unclear. In the current study, overexpression of CD133 increased expression and secretion of IL1ß (IL1B), which activates an autocrine signaling loop that upregulates NF-κB signaling, epithelial-mesenchymal transition (EMT), and cellular invasion. This signaling pathway also induces CXCR4 expression, which in turn is instrumental in imparting an invasive phenotype to these cells. In addition to the autocrine signaling of the CD133 secreted IL1ß, the tumor-associated macrophages (TAM) also produced IL1ß, which further activated this pathway in TICs. The functional significance of the TIC marker CD133 has remained elusive for a very long time; the current study takes us one step closer to understanding how the downstream signaling pathways in these cells regulate the functional properties of TICs.Implications: This study demonstrates the important role of tumor- and macrophage-derived IL1ß stimulation in pancreatic cancer. IL1 signaling is increased in cells with CD133 expression, leading to increased NF-kB activity, EMT induction, and invasion. Increased invasiveness via IL1ß stimulation is mediated by the upregulation of CXCR4 expression. The study highlights the importance of IL1-mediated signaling in TICs. Mol Cancer Res; 16(1); 162-72. ©2017 AACR.

Inhibition of hypoxic response decreases stemness and reduces tumorigenic signaling due to impaired assembly of HIF1 transcription complex in pancreatic cancer.

Pancreatic tumors are renowned for their extremely hypoxic centers, resulting in upregulation of a number of hypoxia mediated signaling pathways including cell proliferation, metabolism and cell survival. Previous studies from our laboratory have shown that Minnelide, a water-soluble pro-drug of triptolide (anti-cancer compound), decreases viability of cancer cells in vitro as well as in vivo. However, its mechanism of action remain elusive. In the current study we evaluated the effect of Minnelide, on hypoxia mediated oncogenic signaling as well as stemness in pancreatic cancer. Minnelide has just completed Phase 1 trial against GI cancers and is currently awaiting Phase 2 trials. Our results showed that upon treatment with triptolide, HIF-1α protein accumulated in pancreatic cancer cells even though hypoxic response was decreased in them. Our studies showed even though HIF-1α is accumulated in the treated cells, there was no decrease in HIF-1 binding to hypoxia response elements. However, the HIF-1 transcriptional activity was significantly reduced owing to depletion of co-activator p300 upon treatment with triptolide. Further, treatment with triptolide resulted in a decreased activity of Sp1 and NF-kB the two major oncogenic signaling pathway in pancreatic cancer along with a decreased tumor initiating cell (TIC) population in pancreatic tumor.

Inhibition of Sp1 prevents ER homeostasis and causes cell death by lysosomal membrane permeabilization in pancreatic cancer.

Endoplasmic reticulum (ER) stress initiates an important mechanism for cell adaptation and survival, named the unfolded protein response (UPR). Severe or chronic/prolonged UPR can breach the threshold for survival and lead to cell death. There is a fundamental gap in knowledge on the molecular mechanism of how chronic ER stress is stimulated and leads to cell death in pancreatic ductal adenocarcinoma (PDAC). Our study shows that downregulating specificity protein 1 (Sp1), a transcription factor that is overexpressed in pancreatic cancer, activates UPR and results in chronic ER stress. In addition, downregulation of Sp1 results in its decreased binding to the ER stress response element present in the promoter region of Grp78, the master regulator of ER stress, thereby preventing homeostasis. We further show that inhibition of Sp1, as well as induction of ER stress, leads to lysosomal membrane permeabilization (LMP), a sustained accumulation of cytosolic calcium, and eventually cell death in pancreatic cancer.

Microenvironment in determining chemo-resistance in pancreatic cancer: Neighborhood matters.

Every year, nearly 300,000 people are diagnosed with pancreatic cancer worldwide, and an equivalent number succumb to this disease. One of the major challenges of pancreatic cancer that contributes to its poor survival rates is the development of resistance to the standard chemotherapy. Heterogeneity of the tumor, the dense fibroblastic stroma, and the aggressive biology of the tumor all contribute to the chemoresistant phenotype. In addition, the acellular components of the tumor microenvironment like hypoxia, stress pathways in the stromal cells, and the cytokines that are secreted by the immune cells, have a definitive role in orchestrating the chemoresistant property of the tumor. In this review, we systematically focus on the role played by the different microenvironmental components in determining chemoresistance of pancreatic tumors.

Inhibition of NF-kappa B pathway leads to deregulation of epithelial-mesenchymal transition and neural invasion in pancreatic cancer.

NF-κB has an essential role in the initiation and progression of pancreatic cancer and specifically mediates the induction of epithelial-mesenchymal transition and invasiveness. In this study, we demonstrate the importance of activated NF-κB signaling in EMT induction, lymphovascular metastasis, and neural invasion. Modulation of NF-κB activity was accomplished through the specific NF-κB inhibitor (BAY 11-7085), triptolide, and Minnelide treatment, as well as overexpression of IKBα repressor and IKK activator plasmids. In the classical lymphovascular metastatic cascade, inhibition of NF-κB decreased the expression of several EMT transcription factors (SNAI1, SNAI2, and ZEB1) and mesenchymal markers (VIM and CDH2) and decreased in vitro invasion, which was rescued by IKK activation. This was further demonstrated in vivo via BAY 11-7085 treatment in a orthotopic model of pancreatic cancer. In vivo NF-κB inhibition decreased tumor volume; decreased tumor EMT gene expression, while restoring cell-cell junctions; and decreasing overall metastasis. Furthermore, we demonstrate the importance of active NF-κB signaling in neural invasion. Triptolide treatment inhibits Nerve Growth Factor (NGF) mediated, neural-tumor co-culture in vitro invasion, and dorsal root ganglia (DRG) neural outgrowth through a disruption in tumor-neural cross talk. In vivo, Minnelide treatment decreased neurotrophin expression, nerve density, and sciatic nerve invasion. Taken together, this study demonstrates the importance of NF-κB signaling in the progression of pancreatic cancer through the modulation of EMT induction, lymphovascular invasion, and neural invasion.

Microenvironment mediated alterations to metabolic pathways confer increased chemo-resistance in CD133+ tumor initiating cells.

Chemoresistance in pancreatic cancer has been attributed to tumor-initiating cells (TICs), a minor sub-population of tumor cells. However, the mechanism of chemo-resistance in these cells is still unclear.In the current study, immunohistochemical analysis of LSL-KrasG12D; LSL-Trp53R172H;PdxCre (KPC) murine tumors indicated that hypoxic regions developed through tumor progression. This hypoxic "niche" correlated with increased CD133+ population that had an increased HIF1A activity. Consistent with this observation, CD133+ cells had increased glucose uptake and activity of glycolytic pathway enzymes compared to CD133- cells. Mass spectrometric analysis (UPLC-TQD) following metabolic labeling of CD133+ cells with [13C]-U6 glucose confirmed this observation. Furthermore, although both populations had functionally active mitochondria, CD133+ cells had low mitochondrial complex I and complex IV activity and lesser accumulation of ROS in response to standard chemotherapeutic compounds like paclitaxel, 5FU and gemcitabine. CD133+ cells also showed increased resistance to all three chemotherapeutic compounds and treatment with Glut1 inhibitor (STF31) reversed this resistance, promoting apoptotic death in these cells similar to CD133- cells.Our study indicates that the altered metabolic profile of CD133+ pancreatic TIC protects them against apoptosis, by reducing accumulation of ROS induced by standard chemotherapeutic agents, thereby confering chemoresistance. Since resistance to existing chemotherapy contributes to the poor prognosis in pancreatic cancer, our study paves the way for identifying novel therapeutic targets for managing chemoresistance and tumor recurrence in pancreatic cancer.

A Novel Immunocompetent Mouse Model of Pancreatic Cancer with Robust Stroma: a Valuable Tool for Preclinical Evaluation of New Therapies.

A valid preclinical tumor model should recapitulate the tumor microenvironment. Immune and stromal components are absent in immunodeficient models of pancreatic cancer. While these components are present in genetically engineered models such as Kras(G12D); Trp53(R172H); Pdx-1Cre (KPC), immense variability in development of invasive disease makes them unsuitable for evaluation of novel therapies. We have generated a novel mouse model of pancreatic cancer by implanting tumor fragments from KPC mice into the pancreas of wild type mice. Three-millimeter tumor pieces from KPC mice were implanted into the pancreas of C57BL/6J mice. Four to eight weeks later, tumors were harvested, and stromal and immune components were evaluated. The efficacy of Minnelide, a novel compound which has been shown to be effective against pancreatic cancer in a number of preclinical murine models, was evaluated. In our model, consistent tumor growth and metastases were observed. Tumors demonstrated intense desmoplasia and leukocytic infiltration which was comparable to that in the genetically engineered KPC model and significantly more than that observed in KPC tumor-derived cell line implantation model. Minnelide treatment resulted in a significant decrease in the tumor weight and volume. This novel model demonstrates a consistent growth rate and tumor-associated mortality and recapitulates the tumor microenvironment. This convenient model is a valuable tool to evaluate novel therapies.

Minnelide effectively eliminates CD133(+) side population in pancreatic cancer.

BACKGROUND: Pancreatic Ductal Adenocarcinoma (PDAC) is a devastating disease hallmarked by limited patient survival. Resistance to chemotherapy, a major cause of treatment failure in PDAC patients, is often attributed to Cancer Stem Cells (CSCs). Pancreatic CSCs are a small subset of quiescent cells within a tumor represented by surface markers like CD133. These cells are responsible not only for tumor recurrence, but also poor prognosis based on their "stem-like" characteristics. At present, conventional therapy is directed towards rapidly dividing PDAC cells and thus fails to target the CSC population. METHODS: MIA PaCa-2, S2-013 and AsPC-1 were treated with 12.5 nM triptolide (12 T cells) for 7 days. The surviving cells were recovered briefly in drug-free growth media and then transferred to Cancer Stem cell Media (CSM). As a control, untreated cells were also transferred to CSM media (CSM). The 12 T and CSM cells were tested for stemness properties using RNA and protein markers. Low numbers of CSM and 12 T cells were implanted subcutaneously in athymic nude mice to study their tumorigenic potential. 12 T and CSM cells were sorted for CD133 expression and assayed for their colony forming ability and sphere forming ability. Invasiveness of 12 T cells, CSM and MIA PaCa-2 were compared using Boyden chamber assays. RESULTS: Treated 12 T cells displayed increased expression of the surface marker CD133 and the drug transporter ABCG2 compared to untreated cells (CSM cells). Both 12 T and CSM cells formed subcutaneous tumors in mice confirming their tumor-initiating properties. When tested for invasion, 12 T cells had increased invasiveness compared to CSM cells. CD133(+) cells in both CSM and 12 T showed greater colony and sphere forming ability compared to CD133(-) cells from each group. Consistent with these data, when injected subcutaneously in mice, CD133(-) cells from CSM or 12 T did not form any tumors whereas CD133(+) cells from both groups showed tumor formation at a very low cell number. Despite pre-exposure to triptolide in 12 T CD133(+) cells, treatment of tumors formed by these cells with Minnelide, a triptolide pro-drug, showed significant tumor regression. CONCLUSION: Our results indicated that triptolide enhanced and enriched the "stemness" in the PDAC cell lines at a low dose of 12.5 nM, but also resulted in the regression of tumors derived from these cells.

Co-deleting Pten with Rb in retinal progenitor cells in mice results in fully penetrant bilateral retinoblastomas.

BACKGROUND: Rb1 is the most frequently mutated gene in the pediatric cancer retinoblastoma, and its loss causes E2F transcription factors to induce proliferation related genes. However, high E2F levels following pRB loss also induce apoptosis-promoting genes as a safeguard mechanism to suppress emergent tumors. Although p53 accumulation and apoptosis induction is believed to be a primary mechanism to eliminate cells with excess E2F activity, p53 deletion doesn't suppress RB/E2F induced apoptosis in vivo in the retina. This prompted us to test the PTEN/PI3K/AKT signaling pathway on RB/E2F apoptosis suppression in vivo, to ascertain if the PI3K pathway may provide a potential avenue for retinoblastoma therapy. METHODS: We developed a mouse model in which Rb1 and Pten were conditionally deleted from retinal progenitor cells using Chx10-Cre, whereas Rbl1 (p107) was constitutively deleted. Pathway components were also tested individually by in vivo electroporation into newborn retinas for an effect on apoptosis and tumor initiation. Mouse retinal tissues were analyzed by immunohistochemistry (IHC) for proliferation, apoptosis, and pathway activation. ShRNAs were used in vitro to assess effects on apoptosis and gene expression. RESULTS: Co-deleting Pten with Rb1 and Rbl1 in mouse retinal progenitor cells (RPCs) causes fully penetrant bilateral retinoblastomas by 30 days and strongly suppresses Rb/E2F-induced apoptosis. In vivo electroporation of constitutively active (ca)-Pik3ca, ca-Akt, or dominant-negative (dn)-Foxo1 into apoptosis prone newborn murine retina with deleted Rb/p107 eliminate Rb/E2F induced apoptosis and induce retinoblastoma emergence. Retinal deletion of Pten activates p-AKT and p-FOXO1 signaling in incipient retinoblastoma. An unbiased shRNA screen focusing on Akt phosphorylation targets identified FOXOs as critical mediators of Rb/E2F induced apoptosis and expression of Bim and p73 pro-apoptotic genes. CONCLUSIONS: These data indicate that we defined a key molecular trigger involving E2F/FOXO functioning to control retinal progenitor cell homeostasis and retinoblastoma tumor initiation. We anticipate that our findings could provide contextual understanding of the proliferation of other progenitor cells, considering the high frequency of co-altered signaling from RB/E2F and PTEN/PI3K/AKT pathways in a wide variety of normal and malignant settings.

CD133 initiates tumors, induces epithelial-mesenchymal transition and increases metastasis in pancreatic cancer.

CD133 has been implicated as a cancer stem cell (CSC) surface marker in several malignancies including pancreatic cancer. However, the functional role of this surface glycoprotein in the cancer stem cell remains elusive. In this study, we determined that CD133 overexpression induced "stemness" properties in MIA-PaCa2 cells along with increased tumorigenicity, tumor progression, and metastasis in vivo. Additionally, CD133 expression induced epithelial-mesenchymal transition (EMT) and increased in vitro invasion. EMT induction and increased invasiveness were mediated by NF-κB activation, as inhibition of NF-κB mitigated these effects. This study showed that CD133 expression contributes to pancreatic cancer "stemness," tumorigenicity, EMT induction, invasion, and metastasis.

CD133+ tumor initiating cells in a syngenic murine model of pancreatic cancer respond to Minnelide.

PURPOSE: Pancreatic adenocarcinoma is the fourth leading cause for cancer-related mortality with a survival rate of less than 5%. Late diagnosis and lack of effective chemotherapeutic regimen contribute to these grim survival statistics. Relapse of any tumor is largely attributed to the presence of tumor-initiating cells (TIC) or cancer stem cells (CSC). These cells are considered as hurdles to cancer therapy as no known chemotherapeutic compound is reported to target them. Thus, there is an urgent need to develop a TIC-targeted therapy for pancreatic cancer. EXPERIMENTAL DESIGN: We isolated CD133(+) cells from a spontaneous pancreatic ductal adenocarcinoma mouse model and studied both surface expression, molecular markers of pancreatic TICs. We also studied tumor initiation properties by implanting low numbers of CD133(+) cells in immune competent mice. Effect of Minnelide, a drug currently under phase I clinical trial, was studied on the tumors derived from the CD133(+) cells. RESULTS: Our study showed for the first time that CD133(+) population demonstrated all the molecular markers for pancreatic TIC. These cells initiated tumors in immunocompetent mouse models and showed increased expression of prosurvival and proinvasive proteins compared to the CD133(-) non-TIC population. Our study further showed that Minnelide was very efficient in downregulating both CD133(-) and CD133(+) population in the tumors, resulting in a 60% decrease in tumor volume compared with the untreated ones. CONCLUSION: As Minnelide is currently under phase I clinical trial, its evaluation in reducing tumor burden by decreasing TIC as well as non-TIC population suggests its potential as an effective therapy.

Dynamic tumor growth patterns in a novel murine model of colorectal cancer.

Colorectal cancer often arises from adenomatous colonic polyps. Polyps can grow and progress to cancer, but may also remain static in size, regress, or resolve. Predicting which polyps progress and which remain benign is difficult. We developed a novel long-lived murine model of colorectal cancer with tumors that can be followed by colonoscopy. Our aim was to assess whether these tumors have similar growth patterns and histologic fates to human colorectal polyps to identify features to aid in risk stratification of colonic tumors. Long-lived Apc(Min/+) mice were treated with dextran sodium sulfate to promote colonic tumorigenesis. Tumor growth patterns were characterized by serial colonoscopy with biopsies obtained for immunohistochemistry and gene expression profiling. Tumors grew, remained static, regressed, or resolved over time with different relative frequencies. Newly developed tumors demonstrated higher rates of growth and resolution than more established tumors that tended to remain static in size. Colonic tumors were hyperplastic lesions (3%), adenomas (73%), intramucosal carcinomas (20%), or adenocarcinomas (3%). Interestingly, the level of ß-catenin was higher in adenomas that became intratumoral carcinomas than those that failed to progress. In addition, differentially expressed genes between adenomas and intramucosal carcinomas were identified. This novel murine model of intestinal tumorigenesis develops colonic tumors that can be monitored by serial colonoscopy, mirror growth patterns seen in human colorectal polyps, and progress to colorectal cancer. Further characterization of cellular and molecular features is needed to determine which features can be used to risk-stratify polyps for progression to colorectal cancer and potentially guide prevention strategies.

Application of a whole-body pharmacokinetic model for targeted radionuclide therapy to NM404 and FLT.

We have previously developed a model that provides relative dosimetry estimates for targeted radionuclide therapy (TRT) agents. The whole-body and tumor pharmacokinetic (PK) parameters of this model can be noninvasively measured with molecular imaging, providing a means of comparing potential TRT agents. Parameter sensitivities and noise will affect the accuracy and precision of the estimated PK values and hence dosimetry estimates. The aim of this work is to apply a PK model for TRT to two agents with different magnitudes of clearance rates, NM404 and FLT, explore parameter sensitivity with respect to time and investigate the effect of noise on parameter precision and accuracy. Twenty-three tumor bearing mice were injected with a 'slow-clearing' agent, (124)I-NM404 (n = 10), or a 'fast-clearing' agent, (18)F-FLT (3'-deoxy-3'-fluorothymidine) (n = 13) and imaged via micro-PET/CT pseudo-dynamically or dynamically, respectively. Regions of interest were drawn within the heart and tumor to create time-concentration curves for blood pool and tumor. PK analysis was performed to estimate the mean and standard error of the central compartment efflux-to-influx ratio (k(12)/k(21)), central elimination rate constant (k(el)), and tumor influx-to-efflux ratio (k(34)/k(43)), as well as the mean and standard deviation of the dosimetry estimates. NM404 and FLT parameter estimation results were used to analyze model accuracy and parameter sensitivity. The accuracy of the experimental sampling schedule was compared to that of an optimal sampling schedule found using Cramer-Rao lower bounds theory. Accuracy was assessed using correlation coefficient, bias and standard error of the estimate normalized to the mean (SEE/mean). The PK parameter estimation of NM404 yielded a central clearance, k(el) (0.009 ± 0.003 h(-1)), normal body retention, k(12)/k(21) (0.69 ± 0.16), tumor retention, k(34)/k(43) (1.44 ± 0.46) and predicted dosimetry, D(tumor) (3.47 ± 1.24 Gy). The PK parameter estimation of FLT yielded a central elimination rate constant, k(el) (0.050 ± 0.025 min(-1)), normal body retention, k(12)/k(21) (2.21 ± 0.62) and tumor retention, k(34)/k(43) (0.65 ± 0.17), and predicted dosimetry, D(tumor) (0.61 ± 0.20 Gy). Compared to experimental sampling, optimal sampling decreases the dosimetry bias and SEE/mean for NM404; however, it increases bias and decreases SEE/mean for FLT. For both NM404 and FLT, central compartment efflux rate constant, k(12), and central compartment influx rate constant, k(21), possess mirroring sensitivities at relatively early time points. The instantaneous concentration in the blood, C(0), was most sensitive at early time points; central elimination, k(el), and tumor efflux, k(43), are most sensitive at later time points. A PK model for TRT was applied to both a slow-clearing, NM404, and a fast-clearing, FLT, agents in a xenograft murine model. NM404 possesses more favorable PK values according to the PK TRT model. The precise and accurate measurement of k(12), k(21), k(el), k(34) and k(43) will translate into improved and precise dosimetry estimations. This work will guide the future use of this PK model for assessing the relative effectiveness of potential TRT agents.