TRAMP mouse models of autochthonous prostate cancer groups (n = 20) were injected intraperitoneally with 10 5 , 10 6 , and 10 7 CFU of Salmonella strain CRC2631 in 100μl PBS. Intraperitoneal (IP) injections of 100μl PBS were performed in one group as a negative control. Survival curves were plotted during the study period ( Fig 1 ). Survival curves indicate no significant change in survival for the TRAMP model over the study period at any injection level ( S1 Fig , S1 Table ). One mouse in the 10 7 group died from combat-associated injuries with cage mates; this death was excluded in the survival curve analysis because we cannot tell if the death was due to combat injuries or tumor burden.

Prostate and prostate-associated tumors were extracted from surviving TRAMP mice at study endpoint. Volumes of prostate-associated tumor masses were measured using calipers ( Fig 2 ). Mice with no visible tumor masses were not measured. Visible tumor mass mice dosed with Salmonella had 29.2% smaller average tumor burdens. Average volume of recovered tumors decreased with increase in Salmonella dosage. Due to the single data point in the control group, this data is qualitative.

Histological grading of prostate tumors in the TRAMP mouse model

Prostates along with any associated tumors were extracted from the surviving TRAMP mouse models and fixed overnight in 10% buffered formalin at 4°C. Two cross sections of the dorsal prostate and any associated tumors were sampled by sectioning, hematoxylin and eosin staining, and grading using previously established criteria [49]. Histological grading of Salmonella-treated prostates ranged from normal tissue to poorly differentiated carcinomas (Fig 3). Comparing tumor stages individually, using Chi-square test for association for PDC and Fisher’s exact test for PHY and WD (because some cell sizes are smaller than 5), The p-values were all non-significant: PDC p-value = 0.1725, PHY p-value = 0.3967, WD p-value = 0.4458. Overall association between the injection groups and tumor progression is not statistically significant (P-value = 0.3314) using Fisher’s exact test. Therefore, Salmonella injections did not significantly inhibit tumor progression in the TRAMP prostate cancer model (Table 2). Histological observation indicated the presence of neuroendocrine-type tumors at the periurethral region in twelve TRAMP prostate samples (example, Fig 4). While neuroendocrine tumors in the TRAMP model have been previously reported to invade the periurethral region, this has previously been reported as always associated with a morphologically identical large tumor arising in the prostate [47]; we only observed large neuroendocrine tumors associated with two of the twelve samples.

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larger image TIFF original image Download: Fig 3. Histology of prostate tumor development in TRAMP mice. Histologic sections of the dorsal lobes of the prostate from transgenic mice stained with hematoxylin and eosin at 40X magnification. Pathologic grades: PIN, prostatic epithelial neoplasia; WD, well-differentiated adenocarcinoma; PHY, phylloides-like; PDC, poorly differentiated neuroendocrine-type carcinoma. Slides: (A) Normal tissue, (B) Hyperplastic tissue, (C) PIN, (D) WD, (E) PHY and (F) PDC (neuroendocrine-type). Observations: (C) Note tufting of epithelial cells, increased mitoses, hyperchromatic nuclei, stratification of nuclei and cribiform structures (arrow). (D) Note neoplastic cells with round nuclei; tumor type is characterized by increased numbers of small glands and thickening of the stroma. (E) Note staghorn luminal patterns of neoplastic cells. (F) Note the high nuclear:cytoplasmic ratio of neoplastic cells, loss of glandular differentiation and marked cell pleomorphism. https://doi.org/10.1371/journal.pone.0160926.g003

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larger image TIFF original image Download: Fig 4. Neuroendocrine-type carcinoma in the periurethral region of a TRAMP mouse. Histologic sections of the periurethral region from a transgenic mouse stained with hematoxylin and eosin (H&E) at 4X (A), 10X (B), 40X (C) and 100X (D). (A): Note discrete tumor (arrow) within the epithelium of the periurethral region. The outline in (A) is the magnified region shown in (C) and (D). https://doi.org/10.1371/journal.pone.0160926.g004

Prostate cancer is still the second-leading cause of cancer-related deaths in men [52]. Advanced cancer treatment still represents medical challenges, as effective cures are still not yet available. While androgen deprivation therapy (ADT) is effective in treating early stages of advanced prostate cancer most patients respond to this treatment initially but their cancers become androgen-independent and most patients become ADT resistant [53, 54].

Furthermore, a surprising emergence of neuroendocrine prostate cancer cells (NEPC) has resulted from androgen deprivation therapy (ADT), which presents new challenges for treatment. While the AR-negative neuroendocrine prostate cancers (NEPC) are rare at the time of initial diagnosis, they can account for 5–30% of advanced prostate cancers promoted by ADT [55].

Effective therapy for advanced stages of androgen-independent prostate cancer is still not yet available. Although progress has been made toward identifying the problems associated with disease progression, it has become clear that there is a need to eradicate the various subpopulations of this heterogeneous disease including the hard-to-treat and oftentimes radiation-resistant cancer stem cell (CSC) population. New therapies are critically needed to target these subpopulations that may require different and combination treatment strategies.

While rigorous and targeted therapies for the various subpopulations of prostate cancer might be developed in the distant future new treatment options aimed at targeting the entire cancer tissue with all subpopulations are currently in development and actively pursued in various laboratories using different approaches. A number of laboratories, including ours, are utilizing attenuated bacteria for targeting and chemotherapeutic activation/delivery (“bacteriotherapy”) of cancers. These approaches are expected to have advantages over surgery and radiotherapy and will also eliminate newly observed cancer stem cell populations that have presented new challenges for treatment of cancer, as the inability to provide new treatment options is still associated with poor prognosis. The cancer stem cell subpopulation is responsible for prostate tumor initiation, recurrence, drug-resistance and metastatic progression. Salmonella targeting significantly reduces the weight of tumors initiated by cancer stem-like cells in several studies [56, 57].

Salmonella have been shown to effectively target and colonize any tumors that can be accessed by the host circulatory system, whether the Salmonella is introduced by intravenous, intraperitoneal, or oral delivery [11, 12]. Salmonella adapted to target tumors for detection have been predicted to colonize and detect tumor masses more than 2000-fold smaller than current tumor detection methods utilizing tomography [19].

Evolved to survive in mammalian hosts, Salmonella has the ability to adapt host membrane vesicles for its own use and can manipulate the placement of the membrane vesicle for replication and infiltration (invasion) of adjacent host cells [27]. As a facultative anaerobe, Salmonella can colonize both the oxygen-rich tumor periphery and anoxic tumor mass [8, 38, 58]. Once the entire tumor is colonized, Salmonella can deliver attached chemotherapies or synthesize molecules including cancer-killing chemotherapeutics, enzymes for activating drugs (prodrugs) at the cancer site [59, 60], transfer and/or express genes to inhibit cancer oncogene expression, or produce immune signaling molecules for cancer immunotherapy [35]. The high infiltration rate of Salmonella makes it superior to current nanoparticle technology that is limited in how far it can penetrate tumor tissue due to the high interstitial pressure characteristic of tumor masses [61]. Novel combination bacteriotherapies including targeted delivery of anti-cancer molecules (carried or synthesized), immunostimulatory peptides or vaccines, enzymes designed to activate prodrugs at tumors, and radiation [62] combination therapies, all concentrated at the tumor site using Salmonella, have been and continue to be actively researched by our laboratory and other laboratories in the field of cancer-targeting bacteriotherapy with increasing levels of success.

The single limitation to Salmonella bacteriotherapy is concern about potential toxicity seen in cancer patients using the VNP20009 Salmonella strain during phase I clinical testing in 2002 [9]. Since that study, efforts have been made by multiple laboratories to reduce toxicity in Salmonella without disrupting its cancer targeting, invasion, and tumor infiltration phenotypes. We engineered a novel, attenuated Salmonella bacteriotherapeutic strain (CRC2631) that is non-toxic and exhibits cancer targeting, invasion, and cancer cell destruction phenotypes [6]. Additionally, we have developed tools to facilitate Salmonella vector delivery of combination chemotherapies in order to increase bacteriotherapeutic effectiveness and reduce the dose of Salmonella needed for clinical effect [63].

The effect of Salmonella monotherapy on prostate tumor progression in an immunocompetent model has not been characterized. In the present study, we examined the effect of weekly administration of our bacteriotherapeutic Salmonella strain (CRC2631) on prostate tumor progression. We used the TRAMP mouse model of prostate cancer that utilizes testosterone-driven expression of the SV40 large and small T-antigen [45] to generate autochthonous primary prostate tumors that eventually develop into poorly differentiated carcinomas with neuroendocrine carcinomas. Tumor progression in the TRAMP model is well documented [49, 50, 64] and provides an excellent model to test prostate tumor progression inhibition of Salmonella monotherapy and combination chemotherapies in an immunocompetent mammalian model that translates well to study prostate cancer progression in human patients.

We have shown that Salmonella CRC2631 injections in the immunocompetent TRAMP model are well tolerated; survival curves of TRAMP mice during the 10–22 week injection period show no significant decrease in survival in TRAMP mice during weekly IP injections of 105−107 CFUs of Salmonella versus control injections of sterile PBS. Secondly, the mean size of visible prostate-associated tumors observed in TRAMP mice decreased when Salmonella was administered; as more Salmonella was administered, the average size of prostate-associated tumors also decreased. Due to the single data point in the PBS control group (Fig 2) we cannot state with confidence that CRC2631 caused significant reduction of tumor size; however this qualitative data in combination with the reduction of TRAMP mice at the final PDC stage of SV40 expression-induced prostate cancer in the 106 group (Table 2) suggests that CRC2631 Salmonella monotherapy is reducing their tumor burden in the models with excess tumor growth and increasing their quality of life. These subtle but promising results with CRC2631 Salmonella monotherapy make the immunocompetent, autochthonous TRAMP prostate cancer progression model an excellent candidate for evaluating combination therapies, including but not limited to inhibitors of the Hedgehog signaling pathway which has previously shown partial inhibition of TRAMP tumor progression [50]. The conclusion that Salmonella bacteriotherapy requires additional carried and/or expressed anti-cancer molecules delivered by tumor-infiltrated Salmonella (combination therapy) is a commonly held opinion by prominent researchers in the field of bacteriotherapy [12, 13, 36, 65].

In humans and mice the normal prostate is composed of stromal and epithelial compartments. The epithelial compartment contains luminal epithelial cells, basal cells and a few scattered neuroendocrine (NE) cells. NE cells have epithelial, neural and endocrine features. They are not evenly distributed in the prostate and are most often found in the periurethral region and verumontanum (colliculus seminalis) in humans [66]. NE cells can also be found in prostate cancer, with increased numbers of these cells in tumors associated with poor prognosis [66].

In humans, the term neuroendocrine differentiation (NED) in prostate cancer (PC) refers to the presence of singly scattered NE cells or cells in small nests in typical prostatic adenocarcinomas [66]. Focal neuroendocrine differentiation is common in human prostatic adenocarcinoma [67]. NED is seen in >30% of prostate cancer and is associated with poor prognosis (high grade and high stage tumors) and androgen independence [68]. About 5–10% of prostatic adenocarcinomas contain large numbers of NE tumor cells, however, pure NE tumors in humans are rare as primary cancers [66].

As in human neuroendocrine PC, neuroendocrine carcinomas in TRAMP mice are associated with rapid growth and metastases and are highly lethal. However, while only a small percentage of human prostate tumors are primary NE cancers, TRAMP mice have a high incidence of neuroendocrine tumors arising in the prostate, which often metastasize to the lymph nodes, lung and liver [67]. In mice, neuroendocrine carcinomas are similar to human neuroendocrine carcinomas in appearance and are characterized by cells with high nuclear:cytoplasmic ratio of neoplastic cells, granular cytoplasm, loss of glandular differentiation and marked cell pleomorphism. They are the most widely metastatic and aggressive mouse prostate cancer [67]. The TRAMP model is therefore also suitable to study NED.

Based on histology analysis and grading, we did not find a significant reduction in tumor progression in the TRAMP model using Salmonella monotherapy. However, we had an unexpected and novel finding. Histological observation indicated the presence of neuroendocrine-type tumors at the periurethral region in twelve TRAMP prostate thick sections. While neuroendocrine tumors in the TRAMP model have been previously reported to invade the periurethral region, this has previously been reported as always associated with a morphologically identical large tumor arising in the prostate [47]; we only observed large neuroendocrine tumors associated with two of the twelve samples. We are quite confident that there are no larger tumors in the rest of the ten prostates with neuroendocrine-type tumors at the periurethral region, which is a novel observation in the TRAMP model. However, we cannot eliminate the possibility that there are small neuroendocrine-type tumors in the prostate because we did not perform serial sections of the ten prostates that did not have visible large tumors. Invasion of neuroendocrine type tumor cells at the periurethral region in the TRAMP model is important, as it demonstrates the utility of this model to study neuroendocrine type tumor cells that have become an important aspect for the treatment of aggressive prostate tumors. As indicated above, in human prostate cancer androgen deprivation therapy (ADT) is commonly used for treatment of prostate cancer, which is associated with promoting the progression of androgen receptor (AR)-positive adenocarcinoma cells (AdPC) to AR negative neuroendocrine prostate cancer (NEPC) through neuroendocrine differentiation (NED). However, treating NEPC is difficult, as no potent drugs are available for this type of cancer progression. We plan to follow up and investigate the effect of Salmonella bacteriotherapy on neuroendocrine type tumor cells.