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Purpose Selection of cytotoxic chemotherapy agents (CCA) based on pre-treatment evaluation of drug sensitivities is a desirable but unmet goal for personalized anticancer treatment strategies. Prior attempts to correlate in vitro Chemo-Response Profiles (CRP) of tumor explants or Circulating Tumor Cells (CTCs) with clinical outcomes have been largely unsuccessful. Methods We present results from a large cohort (n = 5090, three Arms) of patients with various solid organ tumors, where CRP of Circulating Tumor-Associated Cells (C-TACs) was determined against cancer-specific CCA panels to generate a database of 56,466 unique CRP. Results In Arm 1 (n = 230), 93.7% concordance was observed between CRP of C-TACs and concurrently obtained Tumor tissue Derived Cells (TDCs). In arm 2 (n = 2201, pretreated), resistance of C-TACs to ≥ 1 CCA was observed in 79% of cases. In a blinded subset analysis of 143 pretreated patients with radiologically ascertained disease progression, CRP of C-TACs was 87% concordant with in vivo treatment failure. In Arm 3 (n = 2734, therapy naïve), innate resistance of C-TACs to ≥ 1 CCA was observed in 61% of cases. In a blinded subset analysis of 77 therapy naïve patients, in vitro chemo-sensitivity of C-TACs was concordant with radiologically ascertained treatment response to first line CCA in 97% of cases. Conclusion To our knowledge, this is the first expansive and in-depth study demonstrating that real-time CRP of C-TACs is a viable approach for non-invasive assessment of response to CCA in solid organ cancers.

Background Histopathologic examination (HPE) of tumor tissue obtained by invasive biopsy is the standard for cancer diagnosis but is resource‐intensive and has been associated with procedural risks. The authors demonstrate that immunocytochemistry (ICC) profiling of circulating ensembles of tumor‐associated cells (C‐ETACs) can noninvasively provide diagnostic guidance in solid organ cancers. Methods The clinical performance of this approach was tested on blood samples from 30,060 individuals, including 9416 individuals with known cancer; 6725 symptomatic individuals with suspected cancer; and 13,919 asymptomatic individuals with no prior diagnosis of cancer. C‐ETACs were harvested from peripheral blood and profiled by ICC for organ‐specific and subtype‐specific markers relevant to the cancer type. ICC profiles were compared with HPE diagnoses to determine concordance. Results The presence of malignancy was confirmed by the detection of C‐ETACs in 91.8% of the 9416 individuals with previously known cancer. Of the 6725 symptomatic individuals, 6025 were diagnosed with cancer, and 700 were diagnosed with benign conditions; C‐ETACs were detected in 92.6% of samples from the 6025 individuals with cancer. In a subset of 3509 samples, ICC profiling of C‐ETACs for organ‐specific and subtype‐specific markers was concordant with HPE findings in 93.1% of cases. C‐ETACs were undetectable in 95% of samples from the 700 symptomatic individuals who had benign conditions and in 96.3% of samples from the 13,919 asymptomatic individuals. Conclusions C‐ETACs were ubiquitous (>90%) in various cancers and provided diagnostically relevant information in the majority (>90%) of cases. This is the first comprehensive report on the feasibility of ICC profiling of C‐ETACs to provide pan‐cancer diagnostic guidance with accuracy comparable to that of HPE., Immunocytochemistry profiling of circulating ensembles of tumor‐associated cells conveys malignancy status as well as the organ of origin in several cancers, including brain cancer, with high sensitivity, specificity, and accuracy. This approach addresses the long‐unmet need for the noninvasive diagnostic triaging of symptomatic individuals with suspected cancers.

Circulating ensembles of tumor‐associated cells (C‐ETACs) which comprise tumor emboli, immune cells and fibroblasts pose well‐recognized risks of thrombosis and aggressive metastasis. However, the detection, prevalence and characterization of C‐ETACs have been impaired due to methodological difficulties. Our findings show extensive pan‐cancer prevalence of C‐ETACs on a hitherto unreported scale in cancer patients and virtual undetectability in asymptomatic individuals. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of 16,134 subjects including 5,509 patients with epithelial malignancies in various organs and 10,625 asymptomatic individuals with age related higher cancer risk. PBMCs were treated with stabilizing reagents to protect and harvest apoptosis‐resistant C‐ETACs, which are defined as cell clusters comprising at least three EpCAM+ and CK+ cells irrespective of leucocyte common antigen (CD45) status. All asymptomatic individuals underwent screening investigations for malignancy including PAP smear, mammography, low‐dose computed tomography, evaluation of cancer antigen 125, cancer antigen 19‐9, alpha fetoprotein, carcinoembryonic antigen, prostate specific antigen (PSA) levels and clinical examination to identify healthy individuals with no indication of cancer. C‐ETACs were detected in 4,944 (89.8%, 95% CI: 89.0–90.7%) out of 5,509 cases of cancer. C‐ETACs were detected in 255 (3%, 95% CI: 2.7–3.4%) of the 8,493 individuals with no abnormal findings in screening. C‐ETACs were detected in 137 (6.4%, 95% CI: 5.4–7.4%) of the 2,132 asymptomatic individuals with abnormal results in one or more screening tests. Our study shows that heterotypic C‐ETACs are ubiquitous in epithelial cancers irrespective of radiological, metastatic or therapy status. C‐ETACs thus qualify to be a systemic hallmark of cancer., What's new? Circulating Ensembles of Tumor Associated Cells (C‐ETACs) comprised of tumor emboli, immune cells, and fibroblasts pose well‐recognized risks of thrombosis and aggressive metastasis. However, the detection and characterization of C‐ETACs have been impaired by methodological difficulties. Here, the authors have developed a label‐free non‐mechanical process that permits enrichment of viable apoptosis‐resistant C‐ETACs from peripheral blood. They show that heterotypic C‐ETACs are not merely incidental findings in cancer but rather a systemic manifestation of malignancy. C‐ETACs are present in a significant proportion of all solid organ malignancies and are rare in asymptomatic individuals. Monitoring of C‐ETACs could help inform cancer management.

RESILIENT (CTRI/2018/02/011808) was a single arm, open label, phase II/III study to test if label agnostic therapy regimens guided by Encyclopedic Tumor Analysis (ETA) can offer meaningful clinical benefit for patients with relapsed refractory metastatic (r/r-m) malignancies. Patients with advanced refractory solid organ malignancies where disease had progressed following ≥2 lines of systemic treatments were enrolled in the trial. Patients received personalized treatment recommendations based on integrational comprehensive analysis of freshly biopsied tumor tissue and blood. The primary end points were Objective Response Rate (ORR), Progression Free Survival (PFS) and Quality of Life (QoL). Objective Response (Complete Response + Partial Response) was observed in 54 of 126 patients evaluable per protocol (ORR = 42.9%; 95% CI: 34.3%–51.4%, p < 0.0001). At study completion, Disease Control (Complete Response + Partial Response + Stable Disease) was observed in 114 out of 126 patients evaluable per protocol (CBR = 90.5%; 95% CI: 83.9% - 95.0%, p < 0.00001) and Disease Progression in 12 patients. Median duration of follow-up was 138 days (range 31 to 379). Median PFS at study termination was 134 days (range 31 to 379). PFS rate at 90 days and 180 days were 93.9% and 82.5% respectively. The study demonstrated that tumors have latent vulnerabilities that can be identified via integrational multi-analyte investigations such as ETA. This approach identified viable treatment options that could yield meaningful clinical benefit in this cohort of patients with advanced refractory cancers.

Background: Activation of the mTOR signaling pathway is ubiquitous in cancers and a favourable therapeutic target. However, presently approved mTOR inhibitor monotherapies have modest benefits in labeled indications while poor outcomes have been reported for mTOR inhibitor monotherapy when administered in a label-agnostic setting based on univariate molecular indications. The present study aimed to determine whether patient-specific combination regimens with mTOR inhibitors and other anticancer agents selected based on multi-analyte molecular and functional tumor interrogation (ETA: Encyclopedic Tumor Analysis) yields significant treatment response and survival benefits in advanced or refractory solid organ cancers.Methods: We evaluated treatment outcomes in 49 patients diagnosed with unresectable or metastatic solid organ cancers, of whom 3 were therapy naïve and 46 were pre-treated in whom the cancer had progressed on 2 or more prior systemic lines. All patients received mTOR inhibitor in combination with other targeted, endocrine or cytotoxic agents as guided by ETA. Patients were followed-up to determine Objective Response Rate (ORR), Progression Free Survival (PFS) and Overall Survival (OS).Results: The Objective Response Rate (ORR) was 57.1%, the disease Control rate (DCR) was 91.8%, median Progression Free Survival (mPFS) was 4.9 months and median Overall Survival (mOS) was 9.4 months. There were no Grade IV treatment related adverse events (AEs) or any treatment related deaths.Conclusion: Patient-specific combination regimens with mTOR inhibition and other anti-neoplastic agents, when selected based on multi-analyte molecular and functional profiling of the tumor can yield meaningful outcomes in advanced or refractory solid organ cancers.Trial Registration: Details of all trials are available at WHO-ICTRP: https://apps.who.int/trialsearch/. RESILIENT ID CTRI/2018/02/011808. ACTPRO ID CTRI/2018/05/014178. LIQUID IMPACT ID CTRI/2019/02/017548.

We have previously shown that circulating ensembles of tumor-associated cells (C-ETACs) are a systemic hallmark of cancer based on analysis of blood samples from 16,134 individuals including 10,625 asymptomatic individuals and 5,509 diagnosed cases of cancer. C-ETACs were ubiquitously (90%) detected across all cancer types and were rare (3.6%) among the asymptomatic population. Consequently, we hypothesized that asymptomatic individuals with detectable C-ETACs would have a definitively elevated risk of developing cancer as compared with individuals without C-ETACs. In the present manuscript we present 1-year follow-up data of the asymptomatic cohort which shows that C-ETAC positive individuals have a 230-fold (P < 0.00001) higher 1-year cancer risk as compared with individuals where C-ETACs were undetectable. Simultaneously, we also expanded the study to include 4,419 symptomatic individuals, suspected of cancer, prior to undergoing an invasive biopsy for diagnosis. C-ETACs were detected in 4,101 (92.8%) of these 4,419 cases where cancer was eventually confirmed. We conclude that detection of C-ETACs can identify patients at risk of cancer and can be reliably used to stratify asymptomatic individuals with an elevated 1-year risk of cancer. Prevention Relevance: The study evaluated a blood test that can determine if healthy (‘asymptomatic’) individuals without a history of cancer have an increased risk of developing cancer within the next one year. This test can significantly minimize radiological or invasive screening in the majority individuals who do not have any increased risk.

Native tissue structures possess elaborate extracellular matrix (ECM) architectures that inspire the design of fibrous structures in the field of regenerative medicine. We review the literature with respect to the successes and failures, as well as the future promise of biopolymer microthreads as scaffolds to promote endogenous and exogenous tissue regeneration. Biomimetic microthread tissue constructs have been proposed for the functional regeneration of tendon, ligament, skeletal muscle, and ventricular myocardial tissues. To date, biopolymer microthreads have demonstrated promising results as materials to recapitulate the hierarchical structure of simple and complex tissues and well as biochemical signaling cues to direct cell-mediated tissue regeneration. Biopolymer microthreads have also demonstrated exciting potential as a platform technology for the targeted delivery of stem cells and therapeutic molecules. Future studies will focus on the design of microthread-based tissue analogs that strategically integrate growth factors and progenitor cells to temporally direct cell-mediated processes that promote enhanced functional tissue regeneration.

3525 Background: Despite the development of targeted therapy agents and immune checkpoint inhibitors (ICI), cytotoxic anticancer agents remain the mainstay of treatment in several solid organ cancers. However, instances of innate and acquired resistance towards these anticancer agents can lead to treatment failures, which remain undetectable until clinical or radiological manifestation of symptoms suggestive of disease progression. There are presently no viable means or markers to detect or monitor for chemoresistance in real time. Owing to this unmet need, cancer treatment strategies face risks of failure and poor outcomes. Methods: We obtained 15 mL blood from 3,662 patients with various solid organ cancers, of various states and including treatment-naïve and pretreated patients. Circulating Tumor Associated Cells (C-TACs) were enriched and harvested from PBMCs using an epigenetically activating medium that is cytotoxic towards non-malignant epithelial and hematolymphoid cells in blood, but confers survival benefit on apoptosis resistant cells of tumorigenic origin (Circulating Tumor Associated Cells, C-TACs). In a subset of patients, fresh tumor tissue was also obtained from which viable tumor derived cells (TDCs) were obtained. Viable TDCs and C-TACs were treated with a panel of anticancer agents and the surviving cell fraction estimated to determine chemoresistance. Results: Among the 1,325 therapy naïve patients, resistance towards treatment agents was observed in C-TACs from 56.3 % of samples. Among 2,201 pretreated patients’ samples, resistance towards treatment agents was observed in C-TACs from 77.8% of samples. The increased resistance in C-TACs from pretreated patients indicated that the C-TACs had been resistance-educated by prior therapies. In a subset of patients, Chemoresistance profile of C-TACs was observed to be 96.9% concordant with that of tumor derived cells (TDCs) which were concurrently obtained from tumor tissue indicating that C-TACs accurately represent chemo-antecedents of the tumor. Conclusions: Non-invasive chemoresistance profiling of C-TACs is a viable strategy to monitor treatment efficacy in real time. Adoption of this strategy in the clinic will not only guide treatment selection with reduced risk of failure, but will also enable timely therapeutic course correction upon detection of acquired chemoresistance.

3544 Background: There are presently no accepted non-invasive means for detection of cancers in asymptomatic individuals or suspected cases. Radiological and serological investigations, though non-invasive, are not confirmatory and necessitate an invasive biopsy to establish malignant status of suspected findings. Invasive biopsies, in turn, face challenges due to non-representative tumor tissue or in cases where biopsy is impossible or unviable. We hypothesized that Circulating Microtumors (also called as C-ETACs: Circulating Ensembles of Tumor Associated Cells) in peripheral blood are universally present in solid organ cancers and their detection can be linked to malignant status. Methods: We obtained peripheral blood from 14,138 patients with various solid organ cancers as well as 10,625 asymptomatic individuals with age associated elevated risk of cancer. Out of the 14,138 patients with cancer, 25.1 % had local (non-metastatic) disease and 56.4% had metastatic disease as confirmed by radiological evaluation. C-ETACs were enriched and harvested from PBMCs using an epigenetically activating medium that is cytotoxic towards non-malignant epithelial and hematolymphoid cells in blood but confers survival benefit on apoptosis resistant circulating cells of tumorigenic origin and their heterotypic clusters (C-ETACs) in peripheral blood. Viable C-ETACs were identified and further characterized by immunocytochemistry (ICC) profiling. Results: C-ETACs were detected in 89.7% of samples from solid organ cancers irrespective of stage, treatment or present radiological status. C-ETACs were rarely detected (3.0%) in asymptomatic individuals. The asymptomatic individuals where C-ETACs were detected are being followed up periodically so as to enable detection of cancer based on clinical or radiological manifestation of symptoms. Conclusions: C-ETACs are ubiquitous in cancers and unexpected in asymptomatic individuals. Detection of C-ETACs in asymptomatic individuals may be indicative of risk of latent undiagnosed malignancy. The non-invasiveness of this approach makes it convenient for screening large populations for cancer.

Background Refractory cancers pose formidable management challenges. We hypothesized that such malignancies have unexplored vulnerabilities that can be identified using Encyclopedic Tumor Analysis (ETA) and effectively targeted using conventional agents in a label- and organ-agnostic manner to yield treatment benefit. The pan-cancer RESILIENT trial addressed patients with advanced refractory malignancies who were treated with ETA guided treatments regimens without any restrictive eligibility criteria. Methods Molecular Profiling (MP) of patients’ fresh tumor tissue interrogated gene alterations and differentially regulated metabolic pathways to identify molecular targets of approved anticancer agents in a label-agnostic manner. Immunohistochemistry (IHC) identified hormone receptors (HR) that could be targeted with endocrine agents. Chemoresistance and response (CRR) profiling of viable tumor derived cells (TDCs) identified functional vulnerabilities of the tumor against a panel of systemic anticancer agents. Synergistic integration of MP, IHC and CRR datasets (i.e., ETA) generated patient-specific drug priority lists with projected efficacy and safety. Patients who received such ETA-guided treatments were evaluated by PET-CT scans to determine treatment response as well as Objective Response Rate (ORR), Disease Control Rate (DCR) and Progression Free Survival (PFS). Results Among the 200 patients who were screened, 110 patients received ETA-guided treatments and were evaluable for response per protocol. PR was observed in 47 patients (ORR = 42.7%) and 99 patients continued to exhibit PR or SD at study termination (DCR = 90%). Median PFS was 125 days. Median PFS rate at 90 days was 94.0%. No significant therapy related adverse events (AEs) were noted – there were no grade IV AEs or treatment related deaths. Most patients reported stable to improved Quality of Life (QoL) in terms of disease-related symptoms and functional status. Conclusions ETA-guided treatments offer meaningful survival benefits and outperformed available alternatives including checkpoint inhibitors in this heavily pretreated pan-cancer population. Legal entity responsible for the study The authors. Funding Datar Cancer Genetics Limited. Disclosure D. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. D. Akolkar: Full / Part-time employment: Datar Cancer Genetics Limited. V. Datta: Full / Part-time employment: Datar Cancer Genetics Limited. A. Ghaisas: Full / Part-time employment: Datar Cancer Genetics Limited. R. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. H. Singh: Full / Part-time employment: Datar Cancer Genetics Limited. A. Srinivasan: Full / Part-time employment: Datar Cancer Genetics Limited. S. Apurwa: Full / Part-time employment: Datar Cancer Genetics Limited. R. Datar: Shareholder / Stockholder / Stock options, Licensing / Royalties, Officer / Board of Directors: Datar Cancer Genetics Limited. All other authors have declared no conflicts of interest.

A significant challenge in the design and development of biomaterial scaffolds is to incorporate mechanical and biochemical cues to direct organized tissue growth. In this study, we investigated the effect of hepatocyte growth factor (HGF) loaded, crosslinked fibrin (EDCn-HGF) microthread scaffolds on skeletal muscle regeneration in a mouse model of volumetric muscle loss (VML). The rapid, sustained release of HGF significantly enhanced the force production of muscle tissue 60 days after injury, recovering more than 200% of the force output relative to measurements recorded immediately after injury. HGF delivery increased the number of differentiating myoblasts 14 days after injury, and supported an enhanced angiogenic response. The architectural morphology of microthread scaffolds supported the ingrowth of nascent myofibers into the wound site, in contrast to fibrin gel implants which did not support functional regeneration. Together, these data suggest that EDCn-HGF microthreads recapitulate several of the regenerative cues lost in VML injuries, promote remodeling of functional muscle tissue, and enhance the functional regeneration of skeletal muscle. Further, by strategically incorporating specific biochemical factors and precisely tuning the structural and mechanical properties of fibrin microthreads, we have developed a powerful platform technology that may enhance regeneration in other axially aligned tissues.

Skeletal muscle injuries typically result from traumatic incidents such as combat injuries where soft-tissue extremity injuries are present in one of four cases. Further, about 4.5 million reconstructive surgical procedures are performed annually as a result of car accidents, cancer ablation, or cosmetic procedures. These combat- and trauma-induced skeletal muscle injuries are characterized by volumetric muscle loss (VML), which significantly reduces the functionality of the injured muscle. While skeletal muscle has an innate repair mechanism, it is unable to compensate for VML injuries because large amounts of tissue including connective tissue and basement membrane are removed or destroyed. This results in a significant need to develop off-the-shelf biomimetic scaffolds to direct skeletal muscle regeneration. Here, the structure and organization of native skeletal muscle tissue is described in order to reveal clear design parameters that are necessary for scaffolds to mimic in order to successfully regenerate muscular tissue. We review the literature with respect to the materials and methodologies used to develop scaffolds for skeletal muscle tissue regeneration as well as the limitations of these materials. We further discuss the variety of cell sources and different injury models to provide some context for the multiple approaches used to evaluate these scaffold materials. Recent findings are highlighted to address the state of the field and directions are outlined for future strategies, both in scaffold design and in the use of different injury models to evaluate these materials, for regenerating functional skeletal muscle. Statement of Significance Volumetric muscle loss (VML) injuries result from traumatic incidents such as those presented from combat missions, where soft-tissue extremity injuries are represented in one of four cases. These injuries remove or destroy large amounts of skeletal muscle including the basement membrane and connective tissue, removing the structural, mechanical, and biochemical cues that usually direct its repair. This results in a significant need to develop off-the-shelf biomimetic scaffolds to direct skeletal muscle regeneration. In this review, we examine current strategies for the development of scaffold materials designed for skeletal muscle regeneration, highlighting advances and limitations associated with these methodologies. Finally, we identify future approaches to enhance skeletal muscle regeneration.

Large skeletal muscle defects that result in volumetric muscle loss (VML) result in the destruction of the basal lamina, which removes key signaling molecules such as hepatocyte growth factor (HGF) from the wound site, eliminating the endogenous capacity of these injuries to regenerate. We recently showed that HGF-loaded fibrin microthreads increased the force production in muscle tissues after 60 days in a mouse VML model. In this study, we created an in vitro, three-dimensional (3D) microscale outgrowth assay system designed to mimic cell recruitment in vivo, and investigated the effect of HGF-loaded, cross-linked fibrin microthreads on myoblast recruitment to predict the results observed in vivo. This outgrowth assay discretely separated the cellular and molecular functions (migration, proliferation, and chemotaxis) that direct outgrowth from the wound margin, creating a powerful platform to model cell recruitment in axially aligned tissues, such as skeletal muscle. The degree of cross-linking was controlled by pH and microthreads cross-linked using physiologically neutral pH (EDCn) facilitated the release of active HGF; increasing the two-dimensional migration and 3D outgrowth of myoblasts twofold. While HGF adsorbed to uncross-linked microthreads, it did not enhance myoblast migration, possibly due to the low concentrations that were adsorbed. Regardless of the amount of HGF adsorbed on the microthreads, myoblast proliferation increased significantly on stiffer, cross-linked microthreads. Together, the results of these studies show that HGF loaded onto EDCn microthreads supported enhanced myoblast migration and recruitment and suggest that our novel outgrowth assay system is a robust in vitro screening tool that predicts the performance of fibrin microthreads in vivo.

799 Background: Definitive diagnosis of gastrointestinal (GI) malignancies is reliant on histopathological examination of tumor tissue obtained by invasive biopsies. However, invasive biopsies are associated with procedural risks, complications and expenses. A non-invasive technique for diagnosis of GI cancers is presently unavailable. Here we present a non-invasive diagnostic approach for GI cancers based on immunocytochemical (ICC) profiling of Circulating Tumor Associated Cells (C-TAC) enriched from peripheral blood. Methods: We collected 15 mL peripheral blood from 1052 patients with known diagnosis of Ca Oesophagus (244), Ca Stomach (170) and Ca Colorectum (638) and with histopathological information available from prior tissue analysis. CTACs were harvested following negative enrichment. C-TACs were identified by immunostaining with EpCAM and panCK. Deep ICC characterization was carried out in a subset of 203 samples (100 colorectal, 19 Gastric and 84 Esophageal) using organ specific markers. A subset of 19 samples from Gastric and 94 samples Esophageal cancers were profiled for Her2 and PD-L1 status. Results: C-TACs could be identified and enriched in 1012 out of 1052 patients (96.2% overall sensitivity). Immunostaining for organ-specific markers was possible in all 203 (100%) samples. Her2 positivity was observed in 2/19 Gastric and 19/84 Esophageal samples. PD-L1 (22C3) positivity was observed in 5/19 Gastric and 32/84 Esophageal samples while PD-L1 (28-8) positivity was observed in 2/19 Gastric and 16/84 Esophageal samples. Conclusions: Our results show that ICC profiling of C-TACs can provide necessary diagnostic information non-invasively to substitute conventional procedures dependent on tissue extraction. This approach fulfils most clinical decision-making requirements in GI malignancies.

194 Background: Resistance to combination regimens of fluorouracil, oxaliplatin and irinotecan are commonly observed in Colorectal cancers (CRC). There are presently no viable approaches for ‘real-time’ monitoring of innate and acquired chemoresistance. We used a novel method for chemo-interrogation (CI) by harvesting from peripheral blood sufficient numbers of Circulating-Tumor Associated Cells (CTACs) which are defined as apoptosis-resistant cells of tumorigenic origin which are positive for Epithelial Cell Adhesion Molecule (EpCAM) and pan-cytokeratins (pan-CK) irrespective of CD45 status. Methods: Peripheral blood was collected from 110 patients with confirmed diagnosis of CRC, among whom 56 were recently diagnosed and therapy naïve while 54 were pre-treated. Peripheral blood mononuclear cells (PBMCs) were harvested by centrifugation and treated with commercially available stabilizing agents by a proprietary protocol to stabilize apoptosis resistant C-TACs. Surviving C-TACs were confirmed by immunostaining for EpCAM and pan-CK. Harvested C-TACs were treated in vitro with 5-fluorouracil, irinotecan and oxaliplatin and the fraction of surviving cells estimated to determine resistance profiles. Results: Among the 56 cases of recently diagnosed therapy naïve CRC, innate chemoresistance was observed in 37.5%, 47.3% and 41.8% of samples (unique patient-drug combinations) towards 5-fluorouracil, irinotecan and oxaliplatin respectively. Among the 54 cases of previously treated CRC, acquired chemoresistance was observed in 92.6%, 92.6% and 95.5% of samples towards 5-fluorouracil, irinotecan and oxaliplatin respectively. Conclusions: We show for the first time that sufficient C-TACs can be harvested for meaningful CI in newly diagnosed treatment naïve CRC as well as refractory CRCs. Post-treatment chemoresistance being an order of magnitude higher than the untreated cohort indicates that C-TACs in CRC are resistance-educated by previous treatments and can guide treatment strategy.

Purpose A multicenter, randomized phase II trial, RECORD-3, was conducted to compare first-line everolimus followed by sunitinib at progression with the standard sequence of first-line sunitinib followed by everolimus in patients with metastatic renal cell carcinoma. Patients and Methods RECORD-3 used a crossover treatment design. The primary objective was to assess progression-free survival (PFS) noninferiority of first-line everolimus compared with first-line sunitinib. Secondary end points included combined PFS for each sequence, overall survival (OS), and safety. Results Of 471 enrolled patients, 238 were randomly assigned to first-line everolimus followed by sunitinib, and 233 were randomly assigned to first-line sunitinib followed by everolimus. The primary end point was not met; the median PFS was 7.9 months for first-line everolimus and 10.7 months for first-line sunitinib (hazard ratio [HR], 1.4; 95% CI, 1.2 to 1.8). Among patients who discontinued first-line, 108 (45%) crossed over from everolimus to second-line sunitinib, and 99 (43%) crossed over from sunitinib to second-line everolimus. The median combined PFS was 21.1 months for sequential everolimus then sunitinib and was 25.8 months for sequential sunitinib then everolimus (HR, 1.3; 95% CI, 0.9 to 1.7). The median OS was 22.4 months for sequential everolimus and then sunitinib and 32.0 months for sequential sunitinib and then everolimus (HR, 1.2; 95% CI, 0.9 to 1.6). Common treatment-emergent adverse events during first-line everolimus or sunitinib were stomatitis (53% and 57%, respectively), fatigue (45% and 51%, respectively), and diarrhea (38% and 57%, respectively). Conclusion Everolimus did not demonstrate noninferiority compared with sunitinib as a first-line therapy. The trial results support the standard treatment paradigm of first-line sunitinib followed by everolimus at progression.

Background Innate and acquired chemoresistance to anticancer therapies are a well-known phenomenon in Esophageal Squamous Cell Carcinomas (ESCC). There are presently no viable approaches for real-time monitoring of resistance in ESCC. We used a novel method for chemo-interrogation (CI) by harvesting sufficient number of Circulating-Tumor Associated Cells (C-TACs) which are defined as apoptosis-resistant cells of tumorigenic origin and are positive for Epithelial Cell Adhesion Molecule (EpCAM) and pan-cytokeratins (pan-CK) irrespective of CD45 status. Methods Peripheral blood was collected from 80 patients with confirmed diagnosis of ESCC, among whom 52 were recently diagnosed therapy-naive, while 28 were pretreated patients. Peripheral blood mononuclear cells (PBMCs) were harvested by centrifugation and treated with commercially available stabilizing agents, by a proprietary protocol, to stabilize apoptosis resistant C-TACs. Surviving C-TACs were confirmed by immunostaining for EpCAM, pan-CK and CD45. Harvested C-TACs were treated in vitro with a panel of conventional cytotoxic anticancer agents and fraction of surviving cells were estimated to determine resistance profiles. Results Among the 52 cases of recently diagnosed therapy naive ESCC, innate chemoresistance was observed towards platinum agents in 40.8% samples (unique patient-drug combinations), taxanes in 34.6% samples, topoisomerase inhibitors in 42.9% samples and antimetabolites in 34.6% samples. Among the 28 cases of previously treated ESCC, resistance towards previously administered systemic agents was observed in 87% of all samples, which included resistance towards platinum agents in 87.5% samples, taxanes in 82.1% samples, topoisomerase inhibitors in 100% samples and antimetabolites in 85.7% samples, respectively. Conclusions We show for the first time that sufficient C-TACs can be harvested for meaningful CI in newly diagnosed treatment naive ESCC as well as refractory ESCCs. Post-treatment chemoresistance being an order of magnitude higher than the untreated cohort, indicates that C-TACs in ESCC are resistance-educated by previous treatments and can guide treatment strategy in ESCC. Legal entity responsible for the study The authors. Funding Datar Cancer Genetics Limited. Disclosure D. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. D. Akolkar: Full / Part-time employment: Datar Cancer Genetics Limited. V. Datta: Full / Part-time employment: Datar Cancer Genetics Limited. S. Schuster: Full / Part-time employment: Datar Cancer Genetics Limited. C. Sims: Full / Part-time employment: Datar Cancer Genetics Limited. R. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. A. Srinivasan: Full / Part-time employment: Datar Cancer Genetics Limited. S. Khan: Full / Part-time employment: Datar Cancer Genetics Limited. S. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. V. Mhase: Full / Part-time employment: Datar Cancer Genetics Limited. S. Apurwa: Full / Part-time employment: Datar Cancer Genetics Limited. R. Datar: Shareholder / Stockholder / Stock options, Licensing / Royalties, Officer / Board of Directors: Datar Cancer Genetics Limited. All other authors have declared no conflicts of interest.

Background Though mTOR inhibition is considered an attractive strategy for cancer management, anti-mTOR monotherapies have not shown meaningful benefits. We hypothesized that an Encyclopedic Tumor Analysis (ETA) can identify vulnerabilities in the tumor in addition to mTOR activation. We further hypothesized that tandem synergistic targeting of these vulnerabilities using combination of mTOR inhibitors and other systemic anticancer agents in a label- and organ-agnostic manner can improve outcomes in refractory solid organ cancers as compared to mTOR inhibition monotherapy alone. Methods Molecular Profiling (MP) of patients’ fresh tumor tissue interrogated gene alterations and differentially regulated metabolic pathways to identify druggable molecular targets in a label-agnostic manner. Immunohistochemistry (IHC) identified targetable hormone receptors (HR). Chemoresistance and response (CRR) profiling of viable tumor derived cells (TDCs) identified vulnerabilities of the tumor against a panel of systemic anticancer agents. Molecular indications linked to PIK3CA, mTOR, PTEN or TP53 genes were used for selection of mTOR inhibitors. Synergistic integration of MP, IHC and CRR datasets (i.e., ETA) generated patient-specific drug priority lists with projected efficacy and safety. Patients who received such ETA-guided treatments were evaluated by PET-CT scan to determine treatment response. Results Among 41 patients who received combination treatments, 23 patients showed PR (ORR = 56.1%), 16 showed SD (DCR = 95.1%) and progression was observed in 2 patients. One patient who received monotherapy progressed at 27 days. Median PFS was 110 days (range 27 to 592). In the SHIVA trial where patients with mTOR activation (n = 46) received monotherapy with mTOR inhibitor, median PFS of 72 days (range 57 to 100) was reported. No significant therapy–related adverse events were reported in any patient. Most patients reported stable to improved Quality of Life (QoL). Conclusions ETA-guided combination regimens with mTOR inhibitors offer a viable and efficient strategy in advanced refractory malignancies and outperform mTOR inhibitor monotherapy. Legal entity responsible for the study The authors. Funding Datar Cancer Genetics Limited. Disclosure D. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. D. Akolkar: Full / Part-time employment: Datar Cancer Genetics Limited. V. Datta: Full / Part-time employment: Datar Cancer Genetics Limited. A. Ghaisas: Full / Part-time employment: Datar Cancer Genetics Limited. R. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. H. Singh: Full / Part-time employment: Datar Cancer Genetics Limited. A. Srinivasan: Full / Part-time employment: Datar Cancer Genetics Limited. S. Apurwa: Full / Part-time employment: Datar Cancer Genetics Limited. R. Datar: Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: Datar Cancer Genetics Limited. All other authors have declared no conflicts of interest.

Direct reprogramming of a differentiated somatic cell into a developmentally more plastic cell would offer an alternative to applications in regenerative medicine that currently depend on either embryonic stem cells (ESCs), adult stem cells, or induced pluripotent stem cells (iPSCs). Here we report the potential of select Xenopus laevis egg extract fractions, in combination with exogenous fibroblast growth factor-2 (FGF2), to affect life span, morphology, gene expression, protein translation, and cellular localization of OCT4 and NANOG transcription factors, and the developmental potential of human dermal fibroblasts in vitro. A gradual change in morphology is accompanied by translation of embryonic transcription factors and their nuclear localization and a life span exceeding 60 population doublings. Cells acquire the ability to follow adipogenic, neuronal, and osteogenic differentiation under appropriate induction conditions in vitro. Analysis of active extract fractions reveals that Xenopus egg protein and RNAs as well as exogenously supplemented FGF2 are required and sufficient for induction and maintenance of this phenotypic change. Factors so far identified in the active fractions include FGF2 itself, transforming growth factor-β, maskin, and nucleoplasmin. Identification of critical factors needed for reprogramming may allow for nonviral, chemically defined derivation of human-induced multipotent cells that can be maintained by exogenous FGF2.

A significant challenge in the design of biomimetic scaffolds is combining morphologic, mechanical, and biochemical cues into a single construct to promote tissue regeneration. In this study, we analyzed the effects of different crosslinking conditions on fibrin biopolymer microthreads to create morphologic scaffolds with tunable mechanical properties that are designed for directional cell guidance. Fibrin microthreads were crosslinked using carbodiimides in either acidic or neutral buffer, and the mechanical, structural, and biochemical responses of the microthreads were investigated. Crosslinking in the presence of acidic buffer (EDCa) created microthreads that had significantly higher tensile strengths and moduli than all other microthreads, and failed at lower strains than all other microthreads. Microthreads crosslinked in neutral buffer (EDCn) were also significantly stronger and stiffer than uncrosslinked threads and were comparable to contracting muscle in stiffness. Swelling ratios of crosslinked microthreads were significantly different from each other and uncrosslinked controls, suggesting a difference in the internal organization and compaction of the microthreads. Using an in vitro degradation assay, we observed that EDCn microthreads degraded within 24 h, six times slower than uncrosslinked control threads, but EDCa microthreads did not show any significant indication of degradation within the 7-day assay period. Microthreads with higher stiffnesses supported significantly increased attachment of C2C12 cells, as well as increases in cell proliferation without a decrease in cell viability. Taken together, these data demonstrate the ability to create microthreads with tunable mechanical and structural properties that differentially direct cellular functions. Ultimately, we anticipate that we can strategically exploit these properties to promote site-specific tissue regeneration.

The transcription factor NANOG is essential for maintaining pluripotency in embryonic stem cells. We have previously reported the expression of NANOG in adult human fibroblasts; here we present a more thorough investigation into the expression of NANOG in a panel of both differentiated and undifferentiated human cells. We utilize RT-PCR, qRT-PCR, cloning and sequencing, sequence alignment, restriction digestion, immunocytochemistry, Western blotting, and EMSA to investigate expression of NANOG in a variety of somatic, transformed and stem cell phenotypes. RT-PCR and qRT-PCR analysis revealed the presence of NANOG transcripts in all the cell types examined, albeit at magnitudes lower than human embryonic stem cells. Further investigation by single nucleotide polymorphism analysis of expressed transcripts in several cell types detected a NANOG pseudogene, NANOGP8, one of only two NANOG pseudogenes with the potential of encoding a similar size protein to embryonic NANOG (eNANOG). Our analysis demonstrates that although the NANOG protein is detected in nearly all cells examined, expression of the eNANOG and/or NANOGP8 transcript as well as the sub-cellular localization of the protein is cell type-specific. Additionally, smooth muscle cells, which express exclusively NANOGP8, display nuclear localization of NANOG protein, indicating that NANOGP8 is a protein coding gene possibly functioning as a transcription factor. Lastly, all cell types expressing eNANOG and/or NANOGP8 were found to be capable of binding a NANOG consensus sequence in vitro. We conclude that eNANOG is not exclusively expressed in undifferentiated cells and that both eNANOG and NANOGP8 may function as transcription factors in a cell type-specific manner.

International audience; In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category "RNA processing" was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos.

Animals produced from assisted reproductive technologies suffer from developmental abnormalities and early fetal death at a higher frequency than that observed in those produced by natural breeding. These symptoms are reminiscent of imprinting disruptions in the human and mouse, suggesting the possibility of perturbations in the expression of imprinted genes such as biallelic expression or silencing. H19 is one of the imprinted genes first identified in mice and humans, but its sequence and imprinting status have not been determined in cattle. In the present study, we obtained the majority of the bovine H19 gene sequence (approximately 2311 base pairs), identified a single nucleotide polymorphism (SNP) in exon 5 and determined the frequencies of different alleles containing the SNP. Our analysis demonstrated that, in cattle produced by natural breeding, H19 was indeed imprinted as shown by either predominant or exclusive expression of the maternal allele. We also analyzed the imprinting pattern of H19 in organs of four animals produced by somatic cell nuclear transfer that died shortly after birth or had developed abnormalities that necessitated immediate killing at birth. Three out of four cloned animals showed biallelic expression of H19, supporting our hypothesis that imprinting disruption is present in cloned animals that suffered from developmental abnormalities at birth. Examination of the expression of H19 in the offspring of a cloned animal produced by artificial insemination showed that the imprinting pattern in this animal was indistinguishable from those of control animals, suggesting that either imprinting disruptions in cloned animals are corrected through natural reproduction or that they are not present in healthy cloned animals capable of undergoing natural reproduction.

We report enrichment in the efficiency of generating mice transgenic for expression of a human protein in their milk using GFP-mediated preimplantation screening. The transgene array consisted of a functional gene (human alpha-1 antitrypsin under the control of the ovine BLG promoter) linked 5' to a reporter gene (GFP under the control of the murine Oct-4 promoter). GFP expression was detected in blastocysts by fluorescence microscopy and green and nongreen embryos were transferred to recipients in separate groups. In the first experiment, of seven pups that resulted from the transfer of blastocysts expressing GFP, five (71%) were transgenic. The experiment was repeated and of 12 pups that resulted from transfer of GFP-expressing blastocysts, 11 were transgenic (92%). The presence of the reporter cassette used for preimplantation screening did not affect the expression level of alpha-1-antitrypsin in the milk of the transgenic mice. In addition, in a related experiment wherein the GFP reporter gene was co-injected with a second mammary-specific transgene, pINC, no effect on transgene expression was observed. For mice transgenic for the mammary-specific gene alone, expression levels for four different lines were 192, 197, 382, and 415 microg/mL. For mice transgenic for both the mammary-specific transgene and the Oct4-GFP reporter cassette, expression levels for seven different lines were 282, 321, 468, 497, 499, 516, and 806 microg/mL.

Since the first report of live mammals produced by nuclear transfer from a cultured differentiated cell population in 1995 (ref. 1), successful development has been obtained in sheep, cattle, mice and goats using a variety of somatic cell types as nuclear donors. The methodology used for embryo reconstruction in each of these species is essentially similar: diploid donor nuclei have been transplanted into enucleated MII oocytes that are activated on, or after transfer. In sheep and goat pre-activated oocytes have also proved successful as cytoplast recipients. The reconstructed embryos are then cultured and selected embryos transferred to surrogate recipients for development to term. In pigs, nuclear transfer has been significantly less successful; a single piglet was reported after transfer of a blastomere nucleus from a four-cell embryo to an enucleated oocyte; however, no live offspring were obtained in studies using somatic cells such as diploid or mitotic fetal fibroblasts as nuclear donors. The development of embryos reconstructed by nuclear transfer is dependent upon a range of factors. Here we investigate some of these factors and report the successful production of cloned piglets from a cultured adult somatic cell population using a new nuclear transfer procedure.

This chapter will focus primarily on the mechanics and practical details associated with nuclear transfer experiments. However, some of the differences between species as they pertain to technical considerations will be discussed. While structured mostly as an introduction and training guide for the novice, it is hoped that experienced investigators will find some useful information as well. Although the goals of most nuclear transfer experiments vary widely, in most cases the micromanipulation procedures remain constant and are merely a tool used to achieve these goals. Separate sections are devoted to the major topics of tool making, equipment, microscopy, manipulation media, enucleation, cell transfer, and fusion, with an additional section covering piezoelectric nuclear transfer. Consideration of species is given for the most commonly used laboratory and livestock models for experimental embryology. In addition, a section is included on technical improvements such as chemical enucleation and nuclear transfer without micromanipulators to present alternative methods aimed at simplification of some of the technically demanding procedures.

Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-L-lysine (degree of polymerization = 51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 1:1 at a DNA concentration of 50 micrograms ml-1 were transgenic. The transgenic frequency for the pronuclear microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered the in vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.

The polymerase chain reaction (PCR) technique was used to detect a whey acidic protein (WAP) gene and transgene presence in mouse ova cultured to various stages of development after pronuclear microinjection at the one-cell stage. The PCR technique detected an endogenous 442 bp WAP DNA sequence in 78% of one-cell, 88% of two-cell and 94% of four-cell ova, and in 95% of morulae and 97% of blastocysts. The heterologous WAP-human protein C transgene was detected in 88% of one-cell, 88% of two-cell and 44% of four-cell ova, and in 40% of morulae and 29% of blastocysts. For comparison, the integration frequency for transgenic mouse production using the same DNA construct was 22%. After five days ofin vitro culture, embryos that were either developmentally arrested or fragmented were tested for the presence of the transgene. The injected construct was detected in 83% of arrested one-cell, 85% of arrested two-cell, and 85% of fragmented ova. In culture, only 28% of zygotes microinjected with DNA developed to the blastocyst stage compared to 74% of noninjected zygotes, while 63% of zygotes developed to the blastocyst stage after injection of buffer alone. Pronuclear injection of the transgene at concentrations of 1.5, 15 and 50 μg ml−1 resulted in 28, 11 and 9% development to blastocysts and 29, 86 and 88% transgene detection, respectively. Transgene detection was 85, 96 and 97% in degenerate embryos at the respective doses of DNA. These data show that pronuclear microinjection of the transgene is detrimental to subsequent embryonic development. Also, unintegrated copies of the transgene probably exist at least until the blastocyst stage, and thereafter are degraded to the extent that they can no longer be detected by PCR.

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.

The goal of this project is to create an accurate model for skeletal muscle in vitro, with a specific focus to enable mechanical stimulation of the tissue. The project began by creating a list of functions, objectives, and constraints based upon a client needs statement. Conceptual designs were then brainstormed and tested for feasibility in order to select components of final design. The design is currently in assembly and testing phases including testing with skeletal muscle tissue formation.

Large-scale musculoskeletal wounds, such as those seen in trauma injuries, present poor functional healing prognoses. In severe trauma, when the native tissue architecture is destroyed or lost, the regenerative capacity of skeletal muscle is diminished by scar formation. Here we demonstrate that a scaffold system composed of fibrin microthreads can provide an efficient delivery system for cell-based therapies and improve regeneration of a large defect in the tibialis anterior of the mouse. Cell-loaded fibrin microthread bundles implanted into a skeletal muscle resection reduced the overall fibroplasia-associated deposition of collagen in the wound bed and promoted in-growth of new muscle tissue. When fibrin microthreads were seeded with adult human cells, implanted cells contributed to the nascent host tissue architecture by forming skeletal muscle fibers, connective tissue, and PAX7-positive cells. Stable engraftment was observed at 10 weeks postimplant and was accompanied by reduced levels of collagen deposition. Taken together, these data support the design and development of a platform for microthread-based delivery of autologous cells that, when coupled to an in vitro cellular reprogramming process, has the potential to improve healing outcomes in large skeletal muscle wounds.

Induced pluripotent stem (iPS) cells are embryonic stem-like cells produced by forcing expression of a minimal number of key factors in differentiated somatic cells. In many ways, they are indistinguishable from embryonic stem cells in that they can differentiate into any cell type in the body. This development has led to worldwide excitement over the possibility to develop cell-based therapies for a variety of degenerative diseases using cells derived from and thus genetically matched to the patient they are aimed to treat. This chapter reviews the scientific foundation that has led to the ability to create iPS cells and the current methods used to make them, as well as the studies that have been conducted to help decipher the molecular pathways involved. The evolution of steps developed in recent years to improve both the efficiency and the safety of the process for clinical and in vitro diagnostic use is also presented.

Reprogramming of differentiated somatic cells into induced pluripotent stem (iPS) cells has potential for derivation of patient-specific cells for therapy as well as for development of models with which to study disease progression. Derivation of iPS cells from human somatic cells has been achieved by viral transduction of human fibroblasts with early developmental genes. Because forced expression of these genes by viral transduction results in transgene integration with unknown and unpredictable potential mutagenic effects, identification of cell culture conditions that can induce endogenous expression of these genes is desirable. Here we show that primary adult human fibroblasts have basal expression of mRNA for OCT4, SOX2, and NANOG. However, translation of these messages into detectable proteins and their subcellular localization depends on cell culture conditions. Manipulation of oxygen concentration and FGF2 supplementation can modulate expression of some pluripotency related genes at the transcriptional, translational, and cellular localization level. Changing cell culture condition parameters led to expression of REX1, potentiation of expression of LIN28, translation of OCT4, SOX2, and NANOG, and translocation of these transcription factors to the cell nucleus. We also show that culture conditions affect the in vitro lifespan of dermal fibroblasts, nearly doubling the number of population doublings before the cells reach replicative senescence. Our results suggest that it is possible to induce and manipulate endogenous expression of stem cell genes in somatic cells without genetic manipulation, but this short-term induction may not be sufficient for acquisition of true pluripotency. Further investigation of the factors involved in inducing this response could lead to discovery of defined culture conditions capable of altering cell fate in vitro. This would alleviate the need for forced expression by transgenesis, thus eliminating the risk of mutagenic effects due to genetic manipulation.

Bos taurus is a good model for embryo biotechnologies such as nuclear transfer. However, animals produced from these technologies often suffer from large calf syndrome, suggesting fetal growth dysregulation. The imprinted fetal mitogen IGF2 is clustered with H19 and the two genes are co-regulated in humans and mice. Although the allelic expression pattern of IGF2/H19 has been elucidated in agricultural species such as sheep and cattle, the underlying mechanism of their imprinting regulation has not been characterized. Using bisulfite sequencing the methylation status of 44 CpG sites in a CpG rich intergenic region of IGF2/H19 in the liver, brain, lung, kidney and placenta of control calves (produced by conventional breeding). One fragment containing 16 CpG sites was differentially methylated region (DMR), and thus may be important in regulating IGF2/H19 allelic expression. The DMR in tissues from cloned term calves that either died immediately after birth or were sacrificed due to complications shortly thereafter were examined. There were significant variations in the methylation of this DMR in some of the cloned animals compared to the controls. Most of the observed variations tended toward hypomethylation. The hypomethylation of this DMR in the liver and placenta of clones correlates with the previous observation of abnormal, biallelic expression of the H19 allele in those clones [Zhang, S., Kubota, C., Yang, L., Zhang, Y., Page, R., O'Neill, M., Yang, X., Tian, X.C., 2004. Genomic imprinting of H19 in naturally reproduced and cloned cattle. Biol. Reprod.] but not with allelic expression of IGF2 (as determined in this study). These data suggest that this DMR is involved in H19 allelic expression, but that other mechanisms probably regulate the expression of IGF2/H19. Contrary to global hypermethylation observed in cloned embryos, putative imprinting control regions can display hypomethylation trends in specific organs of cloned calves.

Research on, and commercialization of, cloned cattle has been conducted for more than 20 years. Early techniques relied on the physical splitting of embryos or using embryo cells for nuclear transfer to generate cloned animals. Milk and meat from these animals entered into the human food market with no evidence of problems. With the advent of nuclear transfer, which enables the direct transference and preservation of high-value meat- and milk-producing genotypes to offspring, concerns have been raised about whether the products from somatic cell nuclear transfer-produced animals are safe for human consumption. Studies on the biochemical properties of food products from cloned and noncloned animals have thus far not detected any differences. All data to date indicate no significant differences in the measured parameters between animals created by nuclear transfer and normally bred animals. Public acceptance of cloned animal products depends upon forthcoming US Food and Drug Administration approval along with convincing safety data.

Fertilisation by intracytoplasmic sperm injection (ICSI), a technique that bypasses the membrane fusion of the gametes, has been widely used to produce offspring in humans and mice. Success with this technique has lent support to the hypothesis that in mammalian fertilisation, a factor from the sperm, the so-called sperm factor, is responsible for oocyte activation and that the fusion process is not involved in the generation of the hallmark [Ca2+]i signalling seen following fertilisation. However, the success of ICSI has largely eluded large domestic species, such as the bovine, porcine and equine, casting doubt on the current model of oocyte activation at fertilisation in these species. Using Ca2+ imagery and a series of treatments to manipulate the chemical structure of the sperm, we have investigated the early events of oocyte activation in response to ICSI in the bovine. Our results demonstrate, for the first time, that following ICSI, the majority of bovine oocytes are unable to mount [Ca2+]i oscillations, although, in few cases, the initiation of [Ca2+]i oscillations can occur in a manner indistinguishable from in vitro fertilisation. We also show that bull sperm possess a full complement of sperm factor. However, either the release and/or activation of the sperm factor are compromised after ICSI, leading to the delivery of a defective Ca2+ stimulus, which results in premature termination of embryo development.

Publisher Summary This chapter focuses primarily on the mechanics and practical details associated with nuclear transfer experiments. As with various technical procedures, around 75% of the success of cloning experiments is attributed to good manipulation tools. Although there are several commercial sources of micromanipulation tools, most skilled and experienced people still prefer to make their own. The two largest suppliers for pre-made micromanipulation tools are Cook Veterinary Products USA and Humagen Fertility Diagnostics Inc. However, once mastered, custom toolmaking does not take too much time and allows the investigator to tailor the tools on the spot for various experimental situations. Micromanipulation pipettes are fashioned from thin-walled glass capillaries, and some investigators use various types of glass for holding and enucleation pipettes. Equipment required to make micromanipulation tools consists of a pipette puller, microforge, pipette grinder, diamond-tip pencil, and gas burner. A pipette puller is used to provide finely drawn pipettes from capillary glass, and a microforge is used to finish and polish the tips of holding and nuclear transfer pipettes. A pipette grinder is used for making beveled tips optimally, and it should offer a variable speed, a nonbumpy grinding surface, and a way of dispensing water over the surface. A diamond-tip pencil is used for making precise cuts in glass capillaries and for making embryo-moving pipettes, and a gas source with a fine flame is used for putting bends into micromanipulation pipettes.

Litter size of DNA microinjected zygotes is lower than for non-manipulated zygotes. The rate of embryonic and fetal survival in early, mid and late gestation was determined to assess whether DNA integration was responsible for embryonic losses. Also, the effect of including non-microinjected embryos with injected embryos on pregnancy rate and transgenic pup production was determined. In Experiment 1, one-cell embryos from immature CD-1 mice were microinjected with a whey acidic protein promoter-human protein C gene construct. One hour after microinjection embryos were transferred to pseudopregnant recipients (45 transfers of 30 embryos each). Fifteen recipients were sacrificed on day 4, 12 and 18 of gestation and the embryos/fetuses analysed for the transgene. The percentage of embryos or fetuses that were positive for the transgene was not significantly different at any day. However, the number of viable embryos at day 4 was significantly greater than fetuses on days 12 or 18. In addition, a high degree of mosaicism was observed in day 18 fetuses and placentae recovered. In Experiment 2, one-cell embryos from CD-1 mice were microinjected and co-transferred with non-manipulated embryos (C57BL/6). Pregnancy rate and the total number of pups born were improved by addition of non-injected embryos. However, the number of transgenic mice produced was similar whether non-injected embryos were included or not. There were 32.2% (15/46) transgenic pups when 0 non-injected embryos were transferred compared with 15.1% (13/86) transgenic pups when 4 or 8 non-injected embryos were added to the transfers. In summary, a high degree of embryonic and fetal mortality occurs among microinjected embryos. Furthermore, since the percentage of transgenesis did not change throughout pregnancy, DNA integration does not appear to account for all of the embryonic losses. other factor(s) related to the microinjection procedure may be involved in the embryonic and fetal failure of microinjected embryos. Addition of non-injected embryos, although it increased pregnancy rate and the number of pups born from microinjected embryos, actually decreased the number of transgenic pups obtained per pregnancy.

Follicular development and ovulation were examined in superovulated Nubian and Nubian-cross dairy goats following prostaglandin F(2alpha) (PGF(2alpha)) and/or gonadotropin releasing hormone (GnRH) treatment. Estrus was synchronized with Synchromate-B((R)) implants. Superovulation was induced with follicle stimulating hormone (FSH) and augmented with GnRH and/or PGF(2alpha). The PGF(2alpha) treatment was administered on Day 2 of superovulation. Implants were removed from all goats on Day 3 of superovulation. The GnRH treatment was administered 24 h after implant removal. All does were exposed to fertile males for 48 h at the time of GnRH injection. Surgical embryo recovery and ovarian response evaluation were conducted 64 to 78.5 h after implant removal. The number of ovulations was higher with GnRH treatment (18.5 +/- 7; x +/- SEM) than that in the controls (5.3 +/- 4.1; P0.05). There were fewer follicles in the GnRH-treated does than in the untreated does (10.9 +/- 2.9 vs 22.1 +/- 3.2; P0.05). The number of follicles smaller than 4 mm in diameter (5.8 +/- 0.8) did not differ between treatments. The GnRH-treated does had fewer 4- to 8-mm follicles (4.2 +/- 2.0 vs 9.1 +/- 1.6; P0.05) and fewer follicles larger than 8 mm (0.7 +/- 1.4 vs 7.3 +/- 1.6; P0.01) than the controls. Predicted times for 1- and 2-cell embryo recoveries were 68.5 and 73.7 h following implant removal, respectively. This study demonstrates that GnRH is an effective supplement used with FSH superovulation regimens in dairy goats. Moreover, GnRH provides for enhanced early embryo collection for DNA microinjection studies.

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.

Cattle and sheep embryos transferred after in vitro production are often afflicted by large offspring syndrome (LOS), which has been correlated with the presence of serum and/or cell co-culture. Previous research indicates that post-fertilization culture affects blastocyst quality and gene expression, and in vitro oocyte maturation and fertilization impact developmental competence. To dissect the effects of in vitro maturation, fertilization, and culture, we compared the expression profiles of single bovine blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF, n = 15); (2) in vivo maturation, in vivo fertilization, and in vitro culture (IVD, n = 14); and (3) in vivo maturation, fertilization, and development (AI, n = 14). For in vitro culture, the embryos were cultured for 2 days in CR1aa medium with bovine serum albumin (BSA) and then transferred to CR1aa with 10% fetal bovine serum (FBS) with cumulus cells until Day 7, at which time the embryos were vitrified. IVD zygotes were surgically collected from two superovulated Holstein donor cows 24 h post-insemination and cultured in the same system. To conduct expression profiling, total RNA was isolated from individual thawed embryos. The RNA was subjected to three rounds of amplification utilizing a previously adapted and validated T7 linear amplification protocol. Amplified RNA from each embryo and from a standard reference was indirectly labeled with Cy3 or Cy5 by dye swap and hybridized to a custom bovine cDNA microarray containing ~6300 unique genes. After Loess normalization, an ANOVA model (GeneSpring 6.1 and SAS 9.0) was used to identify differentially expressed genes. The P-values were adjusted for multiple comparisons using the false discovery rate approach, and a e2-fold differential criterion was applied. A subset of the differentially expressed genes was verified by real-time RT-PCR. The blastocyst rates for IVF and IVD embryos were 37% and 75%, respectively. There were 305, 365, and 200 genes differentially expressed between the AI and IVD, the IVF and IVD, and the AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and the IVD embryos, making these potential candidates for LOS. There were 61 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology categories 'RNA processing' and 'RNA binding' were over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on embryonic gene expression. This work was supported by USDA grants to X.Y., H.A.L., and X.C.T.

Identification of genes implicated in the biological processes of somatic cell nuclear transfer will improve our understanding of reprogramming events, i.e. the transformation of a lineage-committed cell into a pluripotent one. In addition, the gene expression profile of cloned embryos can help explain the widely reported developmental failures in cloned animals. In this study, we investigated global gene expression profiles of bovine in vitro-fertilized and cloned embryos using Gene Chip Bovine Genome Arrays (Affymetrix, Inc., Santa Clara, CA, USA). For the generation of cloned bovine blastocysts from two adult fibroblast lines (C and D), we employed methods previously proven to generate live offspring and compared these offspring to in vitro-produced blastocysts. Total RNA isolated from groups of 10 blastocysts was amplified by a template-switching PCR. Amplified cDNAs were used to synthesize biotin-labeled antisense RNAs (aRNAs) during and in vitro transcription reaction. Labeled aRNAs were hybridized to microarrays as described by the manufacturer. Experiments were performed in four replicates. Expression data were analyzed using the Significance Analysis of Microarrays (SAM; Tusher et al. 2001 Proc. Natl. Acad. Sci. 98, 5116-5121) procedure and software. Overall, 48.4% and 46% of 23 000 bovine transcripts spotted on the arrays were present in cloned and in in vitro-produced control blastocysts, respectively. The SAM procedure identified 43 genes that changed at least 1.5-fold, with an estimated false discovery rate (FDR) of 20%. Comparison of gene expression between NT embryos produced from two different cell lines and IVF controls with the same criteria revealed 6 (clones from cell line C vs. IVF) and 46 (clones from cell line D vs. IVF) differentially expressed genes. The number of transcripts expressed differentially between the cloned embryos with different donor cell origin was 437. Of the 43 differentially expressed transcripts in cloned blastocysts, 13 have unknown functions and the rest of the genes related to cell structure (tuftelin, desmoplakin), cell cycle/mitosis (Kinesin like 4, katanin, stathmin, PCNA), energy metabolism (lactate dehydrogenase, ATPsynthase, lipid-binding protein, keto acid dehydrogenase E1, metallothionein), and cell signaling (GTP-binding protein1, GTP binding stimulatory protein). Our results indicate that expression profiles of cloned blastocysts could be affected by somatic donor cell.

The high rate of gestational loss in nuclear transfer (NT) pregnancies is a major economic and animal cost, preventing the widespread use of the technique. It is currently not possible to predict which early gestation fetuses will remain viable to term. As placental abnormalties are commonly associated with nonviable fetuses, placental proteins such as pregnancy specific protein (PSP 60, Heyman et al., 2002 Biol. Reprod. 63, 1787–1794; or PSPb Hill et al., 2000 Biol. Reprod. 66, 6–13) have been investigated as possible markers of viable pregnancy. To build upon these studies, we explored the predictive value of Day-35 maternal serum PSPb value to final cloned-calf viability; PSPb profiles throughout pregnancy; and the relationship of maternal plasma estradiol levels to calf viability. Maternal serum was sampled for PSPb at the time of the first ultrasound pregnancy examination (Day 35) to determine fetal number and viability (heartbeat). PSPb values were determined using an RIA by Dr. Garth Sasser (BioTracking, Moscow, ID). Median PSPb values were compared using the Kruskal-Wallis one-way ANOVA test on ranks. PSPb levels from failed single and twin pregnancies were significantly different from those of AI bred controls (P7.5. As PSPb levels at Day 35 were not highly predictive of viable outcome, we detailed the gestational PSPb profile for a different group of cows (n=40) that carried cloned pregnancies beyond Day 90. PSPb was determined at Days 35, 50, 65, and 90, then monthly to Day 240. Although there was no clear statistical significance, PSPb levels from nonviable cloned pregnancies were consistently higher at each time point than for viable pregnancies (14 cows gave birth to live calves) and significantly higher than those of controls. At term, plasma estradiol concentrations were assessed in a group of 5 NT pregnancies. Estradiol was investigated as cloned pregnancies often show a lack of readiness for parturition (e.g. lack of udder development, prolonged gestation, dysmature calves). These 5 pregnancies produced 3 highly viable NT calves: 1 with lowered viability that survived only with a high level of intensive care, and 2 nonviable despite a high level of assistance. In this small group, estradiol levels on the day of birth rose in parallel with viability (viable, 662pg; lowered viability, 170pg; nonviable, 76pg). This is a clinical observation rather than a statistical trend that suggests further investigation may be warranted. In summary, our results suggest maternal PSPb levels have value in identifying the outliers in a population of cloned pregnancies and that term-cloned pregnancies may show abnormally low maternal estradiol concentrations at term. Table 1

The efficiency of production of live calves using somatic cell nuclear transfer was compared among 52 different cell lines representing 43 different genotypes. Cell lines were not genetically modified. Nuclear transfer was performed according to methods described by Cibelli et al., 1998 Science 280, 1256–1258, with modifications. All cells were derived from either explant cultures or enzyme digests of skin biopsies and were cyropreserved and thawed at least 48 hours prior to nuclear transfer. Cells were harvested using either pronase or trypsin at 70 to 90% confluence. Oocytes were either activated prior to fusion or immediately after fusion using ionomycin. The couplets were then cultured in cycloheximide and cytochalsin B for 6 hours. In 36 cases (84%), at least one healthy calf was produced from the initial trial which included transfer to 10 to 20 recipients for each cell line. For 4 of the 7 cases where the initial cell line failed to produce a live calf, a new cell line was derived and the process repeated. In one case where the data are available from the second cell line, 5 live calves were produced from 20 recipients receiving embryos (25%). Results from the other repeated cell lines are pending. For 5 of the different genotypes, nuclear transfer was done at about the same time using two different cell lines, and 4 of these have produced healthy calves from both cell lines. In one case, one cell line produced live calves, and no calves were produced from the other cell line. In total, 167 calves were born, of which 107 are alive and healthy as of this writing (64%), and range in age from 1 to 25 months. There are 86 calves older than 6 months of age and no losses have occurred as calves have aged into early adulthood. Forty-four (26%) of the calves were stillborn, failed to convert to neonatal circulation or were euthanized within 48 hours of birth. The most frequent reason for euthanasia was severe contracture of the limbs (arthrogryposis). This defect occurred even within cell lines that also gave rise to healthy calves, although it was more prevalent with certain cell lines. Other complications among the normal calves born were those of an abnormally large umbilicus or umbilical vessels. In addition, 16 calves were lost after the first 48 hours (13%). Two of these losses were due to accidents and 9 of them were due to complications from umbilical infections. The other 5 calf loses resulted from complications common to young calves such as clostridial infection and ruptured abomasum. Recent improvements in cell line derivation and embryo culture techniques, as well as a higher incidence of natural birth and improved neonatal management, have resulted in healthy calf production efficiencies (from embryos transferred) greater than 30% for 5 independent genotypes. The number of healthy calves produced per embryo transferred was 11 of 20 (55%), 5 of 10 (50%), 5 of 10 (50%), 4 of 11 (36%), and 3 of 10 (30%), for each of these genotypes, respectively. There was no correlation between the efficiency of blastocyst production and pregnancy outcome for the cell lines evaluated in this study. In conclusion, the efficiency of live healthy calf production using somatic cell nuclear transfer remains variable, depending on both the cell line and the genotype. However, efficiencies approaching those obtained using conventional embryo transfer is possible.