Acquired resistance to EGFR tyrosine kinase inhibitors alters the metabolism of human head and neck squamous carcinoma cells and xenograft tumours.

BACKGROUND: Acquired resistance to molecularly targeted therapeutics is a key challenge in personalised cancer medicine, highlighting the need for identifying the underlying mechanisms and early biomarkers of relapse, in order to guide subsequent patient management. METHODS: Here we use human head and neck squamous cell carcinoma (HNSCC) models and nuclear magnetic resonance (NMR) spectroscopy to assess the metabolic changes that follow acquired resistance to EGFR tyrosine kinase inhibitors (TKIs), and which could serve as potential metabolic biomarkers of drug resistance. RESULTS: Comparison of NMR metabolite profiles obtained from control (CAL(S)) and EGFR TKI-resistant (CAL(R)) cells grown as 2D monolayers, 3D spheroids or xenograft tumours in athymic mice revealed a number of differences between the sensitive and drug-resistant models. In particular, we observed elevated levels of glycerophosphocholine (GPC) in CAL(R) relative to CAL(S) monolayers, spheroids and tumours, independent of the growth rate or environment. In addition, there was an increase in alanine, aspartate and creatine+phosphocreatine in resistant spheroids and xenografts, and increased levels of lactate, branched-chain amino acids and a fall in phosphoethanolamine only in xenografts. The xenograft lactate build-up was associated with an increased expression of the glucose transporter GLUT-1, whereas the rise in GPC was attributed to inhibition of GPC phosphodiesterase. Reduced glycerophosphocholine (GPC) and phosphocholine were observed in a second HNSCC model probably indicative of a different drug resistance mechanism. CONCLUSIONS: Our studies reveal metabolic signatures associated not only with acquired EGFR TKI resistance but also growth pattern, microenvironment and contributing mechanisms in HNSCC models. These findings warrant further investigation as metabolic biomarkers of disease relapse in the clinic.

Guidelines for the welfare and use of animals in cancer research.

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

Isolated groin recurrence in vulval squamous cell cancer (VSCC). The importance of node count.

OBJECTIVE: To determine whether there is a node count which can define an adequate inguinofemoral lymphadenectomy (IFL) in primary VSCC. METHODS: A retrospective and prospective review of patients with node negative VSCC who had a full staging IFL. Detection of isolated groin recurrences (IGR) would allow groins with higher risk of groin recurrence to be identified. RESULTS: The median node count of 228 IFLs in 139 patients was eight (0-24). There were six IGR (4.3%). Increased rate of IGR was present in patients with increased age, tumour diameter and depth of invasion, lymphovascular space invasion, unilateral IFL, and moderate/poor tumour grade. In the 138 groins with node counts of eight or greater there were no IGRs compared to six in the patients with either undissected groins or groin node counts less than eight (p = 0.030) Interval to IGR was significantly shorter than other sites of recurrence. Both disease-specific and overall survival were significantly reduced in IGR. CONCLUSIONS: An inadequate IFL is a nodal count of less than eight per groin; both these groins and undissected groins are at increased risk of IGR and should have close surveillance.

Control of lymphatic and hematogenous metastasis of a rat mammary carcinoma by the matrix metalloproteinase inhibitor batimastat (BB-94).

We examined the effects of the synthetic matrix metalloproteinase inhibitor batimastat (BB-94) on lung colonization and spontaneous metastasis of a rat mammary carcinoma, HOSP.1P. This tumor expresses both latent and active forms of the matrix metalloproteinases MMP-2 and MMP-9, although the former, as in human breast cancer, is the most prominent. Administration of batimastat (6 x 30 mg/kg i.p.) inhibited by up to 80% both the number and median weights of HOSP.1P lung colonies following i.v. inoculation of cells. This implies an effect both on seeding efficiency and subsequent tumor development. In spontaneous metastasis assays, limited treatment with batimastat (commencing when s.c. tumors were established and continuing until 5 or 14 days after their surgical removal) significantly inhibited lung metastasis but had little effect on lymphatic metastasis. However, when treatment was initiated 2 days prior to surgery and continued until day 70, 100% of animals survived to day 120 when there was no evidence of metastatic disease. All control animals (n = 25) in two separate experiments died before day 100 with lymphatic, lung, and extrapulmonary metastases. Taken together, these data suggest that lymphatic dissemination by HOSP.1P tumor cells is less susceptible to inhibition by batimastat than vascular invasion, but that long-term treatment can effectively prevent the outgrowth of putative micrometastases in both lymph nodes and lungs, allowing sustained tumor-free survival.

Epidermal growth factor-like ligands differentially up-regulate matrix metalloproteinase 9 in head and neck squamous carcinoma cells.

Head and neck squamous cell carcinomas (HNSCCs) are characterized by a marked propensity for local invasion and dissemination to cervical lymph nodes, with distant metastases developing in 30-40% of cases. Overexpression of the epidermal growth factor receptor (EGFR/c-erbB-1) and/or its ligands and high levels of certain matrix metalloproteinases (MMPs) have been associated with poor prognosis. The aim of this study was to examine the effects of EGFR ligands on gelatinase expression and invasion in HNSCC cell lines. We tested epidermal growth factor (EGF), transforming growth factor alpha, betacellulin, heparin-binding EGF, and amphiregulin and measured expression of gelatinases MMP-9 and MMP-2 in an established squamous carcinoma cell line (Detroit-562) and in two cell lines newly derived from patients with head and neck cancers (SIHN-005A and SIHN-006). Incubation of the cell lines with EGF-like ligands up-regulated MMP-9 (but not MMP-2) expression as measured by semiquantitative reverse transcription-PCR in a dose-dependent manner, with the effects being most marked in cells with high EGFR levels and undetectable in cells with low levels. Maximum stimulation was obtained in a concentration range of 10-100 nM. In addition, we confirmed by zymography that gelatinolytic activity consistent with MMP-9 (Mr 92,000) was up-regulated in parallel with increases in gene expression. Betacellulin (which binds both to EGFR and c-erbB-4 receptors) consistently increased MMP-9 expression and activation to a significantly greater degree than the other four ligands when tested at equimolar concentrations. In parallel with MMP-9 up-regulation, all EGF-like ligands increased tumor cell invasion through Matrigel in in vitro Transwell assays. These activities were independent of ligand effects on cell proliferation. Antagonist (ICR62) or agonist (ICR9) anti-EGFR monoclonal antibodies, respectively, inhibited or potentiated MMP-9 activity and tumor cell invasion induced by all ligands. Furthermore, a monoclonal antibody that neutralizes MMP-9 activity (Abl) also inhibited ligand-induced invasion of HNSCC. We confirmed that tumor cell lines used in these studies (and a larger series not reported here) generally expressed multiple c-erbB receptors and ligands. These results indicate that autocrine or paracrine signaling through EGFR potentiates the invasive potential of HNSCC via the selective up-regulation and activation of MMP-9. Furthermore, ligands such as betacellulin (which is commonly expressed in HNSCC), which can bind to and activate other c-erbB receptors, may be especially potent in this regard.

Radioimmunotherapy of breast cancer xenografts with monoclonal antibody ICR12 against c-erbB2 p185: comparison of iodogen and N-succinimidyl 4-methyl-3-(tri-n-butylstannyl)benzoate radioiodination methods.

C-erbB2 p185 is a proto-oncogene product expressed in 25-30% of human invasive breast cancers that is associated with poor prognosis and resistance to endocrine therapy and chemotherapy. It is minimally expressed in normal adult tissues (M. F. Press et al., Oncogene, 5: 953-962, 1990). For this reason, it is an attractive target for radioimmunotherapy and other antibody-directed therapies. ICR12 is a rat IgG2a monoclonal antibody directed against a protein epitope of the external domain of the c-erbB2 p185. We performed experiments to optimize the direct iodination of ICR12 with 131I using the IodoGen method, and we found impairment of immunoreactive fraction with increasing specific activity. N-Succinimidyl 4-methyl-3-(tri-n-butylstannyl)benzoate (MATE) is a tin ester that can be radioiodinated easily and then coupled to the epsilon-amino group of lysine residues. This method has been shown to have improved uptake in tumors compared with antibody labeled by direct iodination (P. K. Garg et al., Nucl. Med. Biol., 20: 379-387, 1993). ICR12 could be labeled up to 16 mCi/mg by this technique without loss of immunoreactive fraction. Whole-body retention of MATE-labeled ICR12 was less than IodoGen (P < 0.0001). Radioimmunotherapy experiments in athymic mice bearing established MDA MB 361 human breast cancer xenografts showed growth inhibition for > 24 days at a dose of 600 microCi/mouse (P < 0.0001) when labeled by the IodoGen technique, and 12 days using the MATE method (P < 0.0001).

Regression of established breast carcinoma xenografts with antibody-directed enzyme prodrug therapy against c-erbB2 p185.

The enzyme carboxypeptidase G2 (CPG2) was conjugated to the rat IgG2a monoclonal antibody (mAb) ICR12, which recognizes the external domain of the human c-erbB2 protooncogene product. The conjugate retained antigen-binding and enzyme activity. Radiolabeled conjugate localized efficiently and stably to MDA MB 361 breast carcinoma xenografts, which overexpress the c-erbB2 gene product p185. Radiotracer determinations and plasma enzyme activity studies in nu/nu mice gave conjugate blood clearance rate half-lives of approximately 4 days. In separate antibody-directed enzyme prodrug therapy regimes, one dose of the 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid prodrug was administered to nu/nu mice bearing established MDA MB 361 tumors (mean volume, 170-260 mm3). In mice which had received ICR12-CPG2 12-14 days previously, sustained dose-dependent tumor stasis or regressions were effected, which in some cases persisted throughout observation periods of up to 90 days. In control mice which had received the isotype-matched irrelevant mAb ICR16-CPG2 conjugate, tumors grew progressively, as did those in mice treated with prodrug alone, or treated simultaneously with ICR12-CPG2 and prodrug at the maximum tolerated dose. Control chemotherapy with conventional drugs proved toxic and induced only minimal growth delays.

Correlation of plasma and serum vascular endothelial growth factor levels with platelet count in colorectal cancer: clinical evidence of platelet scavenging?

Most studies measuring circulating vascular endothelial growth factor (VEGF) have sampled serum rather than plasma. There has been much debate whether the collection of sera (which causes the activation of platelets and VEGF release) is a true reflection of tumor angiogenic activity or whether platelets act as scavengers of VEGF. Addressing this issue, we measured serum and plasma VEGF, before and after colorectal resection, with reference to platelet counts. Serum and plasma samples were collected from 116 colorectal cancer (CRC) and 116 control patients. Ninety CRC and 32 benign resections were performed. Both plasma and serum VEGF were significantly higher in CRC patients (18.5 and 327 pg/ml, respectively) compared with controls (9.0 and 151.5 pg/ml, respectively; P < 0.0001). Paired serum and plasma VEGF measurements correlated in both CRC (r = 0.56) and control patients (r = 0.73; P < 0.0001). Serum and plasma VEGF levels correlated with platelet count in CRC patients (r = 0.58 and 0.44, respectively) but not in controls. Plasma and serum VEGF levels, and VEGF concentration per platelet, increased with advancing disease stage. The correlation of serum and plasma VEGF with platelet counts in CRC but not in benign disease may be attributable to the scavenging of VEGF from the tumor source by platelets, with plasma levels reflecting free circulating VEGF in equilibrium with platelet levels. VEGF levels in citrated plasma are low and lie close to the limits of ELISA sensitivity. We recommend that a standardized measurement of serum VEGF--normalized by the patient's platelet count to give a value of serum VEGF per platelet--be adopted.

VEGF-A, VEGF-C, and VEGF-D in colorectal cancer progression.

We aimed to assess the relationship of the angiogenic cytokines VEGF-A, VEGF-C, and VEGF-D and their receptors VEGFR-2 and VEGFR-3 in the adenoma-carcinoma sequence and in metastatic spread of colorectal cancer (CRC). mRNA expression levels were measured using semi-quantitative reverse transcription polymerase chain reaction in 70 CRC (35 with paired mucosae) and 20 adenomatous polyps. Immunohistochemistry and ELISA assessed protein expression. VEGF-D mRNA expression was significantly lower in both polyps and CRCs compared with normal mucosa (P=.0002 and.002, respectively), whereas VEGF-A and VEGF-C were significantly raised in CRCs (P=.006 and.004, respectively), but not polyps (P=.22 and P=.5, respectively). Receptor expression was similar in tumor tissue and normal mucosae. Tumors with lymph node metastases had significantly higher levels of VEGF-A compared with non-metastatic tumors (P=.043). There was no association between VEGF-C or VEGF-D and lymphatic spread. The decrease in VEGF-D occurring in polyps and carcinomas may allow the higher levels of VEGF-A and VEGF-C to bind more readily to the VEGF receptors, and produce the angiogenic switch required for tumor growth. Increased expression of VEGF-A within CRCs was associated with lymphatic metastases, and therefore, this member of the VEGF family may be the most important in determining metastatic spread.